Anti-fap antibodies and methods of use

ABSTRACT

The invention provides antibodies against Fibroblast Activation Protein (FAP) and methods of using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/205743, filed on Aug. 9, 2011, which claims the benefit of EuropeanPatent Application No. 10172842.6, filed on Aug. 13, 2010, thedisclosures of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to antibodies specific for FibroblastActivation Protein (FAP). In addition, the invention relates topolynucleotides encoding such antibodies, and vectors and host cellscomprising such polynucleotides. The invention further relates tomethods for producing the antibodies and methods of using them in thetreatment of disease.

BACKGROUND

Fibroblast Activation Protein (FAP) and Anti-FAP Antibodies

Human Fibroblast Activation Protein (FAP; GenBank Accession NumberAAC51668), also known as Seprase, is a 170 kDa integral membrane serinepeptidase (EC 3.4.21.B28). Together with dipeptidyl peptidase IV (alsoknown as CD26; GenBank Accession Number P27487), a closely relatedcell-surface enzyme, and other peptidases, FAP belongs to the dipeptidylpeptidase IV family (Yu et al., FEBS J 277, 1126-1144 (2010)). It is ahomodimer containing two N-glycosylated subunits with a large C-terminalextracellular domain, in which the enzyme's catalytic domain is located(Scanlan et al., Proc Natl Acad Sci USA 91, 5657-5661 (1994)). FAP, inits glycosylated form, has both post-prolyl dipeptidyl peptidase andgelatinase activities (Sun et al., Protein Expr Purif 24, 274-281(2002)).

Human FAP was originally identified in cultured fibroblasts using themonoclonal antibody (mAb) F19 (described in WO 93/05804, ATCC Number HB8269). Homologues of the protein were found in several species,including mice (Niedermeyer et al., Int J Cancer 71, 383-389 (1997),Niedermeyer et al., Eur J Biochem 254, 650-654 (1998); GenBank AccessionNumber AAH19190). FAP has a unique tissue distribution: its expressionwas found to be highly upregulated on reactive stromal fibroblasts ofmore than 90% of all primary and metastatic epithelial tumors, includinglung, colorectal, bladder, ovarian and breast carcinomas, while it isgenerally absent from normal adult tissues (Rettig et al., Proc NatlAcad Sci USA 85, 3110-3114 (1988); Garin-Chesa et al., Proc Natl AcadSci USA 87, 7235-7239 (1990)). Subsequent reports showed that FAP is notonly expressed in stromal fibroblasts but also in some types ofmalignant cells of epithelial origin, and that FAP expression directlycorrelates with the malignant phenotype (Jin et al., Anticancer Res 23,3195-3198 (2003)).

Due to its expression in many common cancers and its restrictedexpression in normal tissues, FAP has been considered a promisingantigenic target for imaging, diagnosis and therapy of a variety ofcarcinomas. Thus, multiple monoclonal antibodies have been raisedagainst FAP for research, diagnostic and therapeutic purposes.

Sibrotuzumab/BIBH1, a humanized version of the F19 antibody thatspecifically binds to human FAP (described in WO 99/57151), and furtherhumanized or fully human antibodies against the FAP antigen with F19epitope specificity (described in Mersmann et al., Int J Cancer 92,240-248 (2001); Schmidt et al., Eur J Biochem 268, 1730-1738 (2001); WO01/68708)) were developed. The OS4 antibody is another humanized(CDR-grafted) version of the F19 antibody (Wüest et al., J Biotech 92,159-168 (2001), while scFv 33 and scFv 36 have a different bindingspecificity from F19 and are cross-reactive for the human and mouse FAPprotein (Brocks et al., Mol Med 7, 461-469 (2001)). More recently, othermurine anti-FAP antibodies, as well as chimeric and humanized versionsthereof, were developed (WO 2007/077173, Ostermann et al., Clin CancerRes 14, 4584-4592 (2008)).

Proteases in the tumor stroma, through proteolytic degradation ofextracellular matrix (ECM) components, facilitate processes such asangiogenesis and/or tumor cell migration. Moreover, the tumor stromaplays an important role in nutrient and oxygen supply of tumors, as wellas in tumor invasion and metastasis. These essential functions make itnot only a diagnostic but also a potential therapeutic target.

Evidence for the feasibility of the concept of tumor stroma targeting invivo using anti-FAP antibodies was obtained in a phase I clinical studywith ¹³¹iodine-lableled F19 antibody, which demonstrated specificenrichment of the antibody in the tumors and detection of metastases(Welt et al., J Clin Oncol 12, 1193-1203 (1994). Similarly, a phase Istudy with sibrotuzumab demonstrated specific tumor accumulation of the¹³¹I-labeled antibody (Scott et al., Clin Cancer Res 9, 1639-1647(2003)). An early phase II trial of unconjugated sibrotuzumab inpatients with metastatic colorectal cancer, however, was discontinueddue to the lack of efficacy of the antibody in inhibiting tumorprogression (Hofheinz et al., Onkologie 26, 44-48 (2003)). Also a morerecently developed anti-FAP antibody failed to show anti-tumor effectsin vivo in unconjugated form (WO 2007/077173).

Thus, there remains a need for enhanced therapeutic approaches,including antibodies with improved efficacy, targeting FAP for thetreatment of cancers.

Antibody Glycosylation

The oligosaccharide component can significantly affect propertiesrelevant to the efficacy of a therapeutic glycoprotein, includingphysical stability, resistance to protease attack, interactions with theimmune system, pharmacokinetics, and specific biological activity. Suchproperties may depend not only on the presence or absence, but also onthe specific structures, of oligosaccharides. Some generalizationsbetween oligosaccharide structure and glycoprotein function can be made.For example, certain oligosaccharide structures mediate rapid clearanceof the glycoprotein from the bloodstream through interactions withspecific carbohydrate binding proteins, while others can be bound byantibodies and trigger undesired immune reactions (Jenkins et al.,Nature Biotechnol 14, 975-81 (1996)).

IgG1 type antibodies, the most commonly used antibodies in cancerimmunotherapy, are glycoproteins that have a conserved N-linkedglycosylation site at Asn 297 in each CH2 domain. The two complexbiantennary oligosaccharides attached to Asn 297 are buried between theCH2 domains, forming extensive contacts with the polypeptide backbone,and their presence is essential for the antibody to mediate effectorfunctions such as antibody dependent cell-mediated cytotoxicity (ADCC)(Lifely et al., Glycobiology 5, 813-822 (1995); Jefferis et al., ImmunolRev 163, 59-76 (1998); Wright and Morrison, Trends Biotechnol 15, 26-32(1997)). Protein engineering studies have shown that FcγRs interact withthe lower hinge region of the IgG CH2 domain. Lund et al., J. Immunol.157:4963-69 (1996). However, FcγR binding also requires the presence ofthe oligosaccharides in the CH2 region. Lund et al., J. Immunol.157:4963-69 (1996); Wright and Morrison, Trends Biotech. 15:26-31(1997), suggesting that either oligosaccharide and polypeptide bothdirectly contribute to the interaction site or that the oligosaccharideis required to maintain an active CH2 polypeptide conformation.Modification of the oligosaccharide structure can therefore be exploredas a means to increase the affinity of the interaction between IgG1 andFcγR, and to increase ADCC activity of IgG1s.

A way to obtain large increases in the potency of monoclonal antibodies,is to enhance their natural, cell-mediated effector functions byengineering their oligosaccharide component as described in Umaña etal., Nat Biotechnol 17, 176-180 (1999) and U.S. Pat. No. 6,602,684 (WO99/54342), the contents of which are hereby incorporated by reference intheir entirety. Umaña et al. showed that overexpression ofβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), aglycosyltransferase catalyzing the formation of bisectedoligosaccharides, in Chinese hamster ovary (CHO) cells significantlyincreases the in vitro ADCC activity of antibodies produced in thosecells. Overexpression of GnTIII in production cell lines leads toantibodies enriched in bisected oligosaccharides, which are generallyalso non-fucosylated and of the hybrid type. If in addition to GnTIII,mannosidase II (ManII) is overexpressed in production cell lines,antibodies enriched in bisected, non-fucosylated oligosaccharides of thecomplex type are obtained (Ferrara et al., Biotechn Bioeng 93, 851-861(2006)). Both types of antibodies show strongly enhanced ADCC, ascompared to antibodies with unmodified glycans, but only antibodies inwhich the majority of the N-glycans are of the complex type are able toinduce significant complement-dependent cytotoxicity (Ferrara et al.,Biotechn Bioeng 93, 851-861 (2006)). Alterations in the composition ofthe Asn 297 carbohydrate or its elimination also affect binding of theantibody Fc-domain to Fcγ-receptor (FcγR) and complement C1q protein,which is important for ADCC and CDC, respectively (Umaña et al., NatBiotechnol 17, 176-180 (1999); Davies et al., Biotechnol Bioeng 74,288-294 (2001); Mimura et al., J Biol Chem 276, 45539-45547 (2001);Radaev et al., J Biol Chem 276, 16478-16483 (2001); Shields et al., JBiol Chem 276, 6591-6604 (2001); Shields et al., J Biol Chem 277,26733-26740 (2002); Simmons et al., J Immunol Methods 263, 133-147(2002)).

BRIEF SUMMARY OF THE INVENTION

The present invention provides antibodies that specifically bind toFibroblast Activation Protein (FAP), having a high affinity and/orenhanced effector function.

In one aspect, the invention is directed to an antibody thatspecifically binds FAP, comprising at least one (i.e. one, two, three,four, five or six) of the complementarity determining regions (CDRs) setforth in SEQ ID NOs 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65,67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101,103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129,131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157,159, 161, 163, 165, 167, 169, 171, 173, 175 and 177. In one embodiment,the antibody comprises three heavy chain CDRs (i.e. HCDR1, HCDR2, andHCDR3) and/or three light chain CDRs (i.e. LCDR1, LCDR2, and LCDR3)selected from SEQ ID NOs 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99,101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127,129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155,157, 159, 161, 163, 165, 167, 169, 171, 173, 175 and 177. In a morespecific embodiment, the antibody comprises an antibody heavy chainvariable region and/or an antibody light chain variable region,particularly both a heavy and light chain variable region, selected fromthe heavy and light chain variable region sequences set forth in SEQ IDNOs 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217,219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245,247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 267, 269, 271, 273,275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 301,303, 305, 307, 309 and 311.

In a particular embodiment, the invention is directed to an antibodythat specifically binds to FAP, wherein the antibody comprises at leastone heavy or light chain complementarity determining region (CDR)selected from the group of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO:27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ IDNO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55,SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO:65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ IDNO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93,SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO:103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO:121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO:139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO:157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO:175, and SEQ ID NO: 177, or a combination thereof.

In one particular embodiment, the invention is directed to an antibodywhich comprises a heavy chain variable region comprising (a) a heavychain CDR1 selected from the group of SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, and SEQ ID NO: 33; (b) a heavychain CDR2 selected from the group of SEQ ID NO: 35, SEQ ID NO: 37, SEQID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47,SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO:57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ IDNO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85,SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO:95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ IDNO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113,SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ IDNO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131,and SEQ ID NO: 133; and (c) a heavy chain CDR3 selected from the groupof SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, and SEQ ID NO: 141.

In a particular embodiment, the invention is directed to an antibodywhich comprises a light chain variable region comprising (a) a lightchain CDR1 selected from the group of SEQ ID NO: 143, SEQ ID NO: 145,SEQ ID NO: 147, and SEQ ID NO: 149; (b) a light chain CDR2 selected fromthe group of SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO:157, SEQ ID NO: 159, and SEQ ID NO: 161; and (c) a light chain CDR3selected from the group of SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, andSEQ ID NO: 177.

In a particular embodiment, the invention is directed to an antibodywhich comprises a heavy chain variable region comprising (a) a heavychain CDR1 selected from the group of SEQ ID NO: 3, SEQ ID NO: 5, SEQ IDNO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ IDNO: 17, SEQ ID NO: 19, SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, and SEQ ID NO: 33; (b) a heavychain CDR2 selected from the group of SEQ ID NO: 35, SEQ ID NO: 37, SEQID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47,SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO:57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ IDNO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85,SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO:95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ IDNO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113,SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ IDNO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131,and SEQ ID NO: 133; and (c) a heavy chain CDR3 selected from the groupof SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, and SEQ ID NO: 141;and a light chain variable region comprising (d) a light chain CDR1selected from the group of SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO:147, and SEQ ID NO: 149; (e) a light chain CDR2 selected from the groupof SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQID NO: 159, and SEQ ID NO: 161; and (f) a light chain CDR3 selected fromthe group of SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO:169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, and SEQ ID NO: 177.In one embodiment, the antibody comprises at least one heavy or lightchain CDR which is not a CDR selected from the group of SEQ ID NO: 3,SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO:25, SEQ ID NO: 27, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ IDNO: 41, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO:135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:171, SEQ ID NO: 173, and SEQ ID NO: 175.

In a particular embodiment, the invention is directed to an antibodywhich comprises a heavy chain variable region comprising an amino acidsequence selected from the group of: SEQ ID NO: 197, SEQ ID NO: 201, SEQID NO: 203, SEQ ID NO: 207, SEQ ID NO: 211, SEQ ID NO: 215, SEQ ID NO:219, SEQ ID NO: 223, SEQ ID NO: 227, SEQ ID NO: 231, SEQ ID NO: 235, SEQID NO: 239, SEQ ID NO: 243, SEQ ID NO: 247, SEQ ID NO: 251, SEQ ID NO:255, SEQ ID NO: 259, SEQ ID NO: 263, SEQ ID NO: 267, SEQ ID NO: 271, SEQID NO: 275, SEQ ID NO: 279, SEQ ID NO: 283, SEQ ID NO: 287, SEQ ID NO:291, SEQ ID NO: 295, SEQ ID NO: 299, SEQ ID NO: 303, SEQ ID NO: 307, andSEQ ID NO: 311.

In a particular embodiment, the invention is directed to an antibodywhich comprises a light chain variable region comprising an amino acidsequence selected from the group of: SEQ ID NO: 193, SEQ ID NO: 195, SEQID NO: 199, SEQ ID NO: 205, SEQ ID NO: 209, SEQ ID NO: 213, SEQ ID NO:217, SEQ ID NO: 221, SEQ ID NO: 225, SEQ ID NO: 229, SEQ ID NO: 233, SEQID NO: 237, SEQ ID NO: 241, SEQ ID NO: 245, SEQ ID NO: 249, SEQ ID NO:253, SEQ ID NO: 257, SEQ ID NO: 261, SEQ ID NO: 265, SEQ ID NO: 269, SEQID NO: 273, SEQ ID NO: 277, SEQ ID NO: 281, SEQ ID NO: 285, SEQ ID NO:289, SEQ ID NO: 293, SEQ ID NO: 297, SEQ ID NO: 301, SEQ ID NO: 305, andSEQ ID NO: 309.

In a particular embodiment, the invention is directed to an antibodywhich comprises a heavy chain variable region comprising an amino acidsequence selected from the group of: SEQ ID NO: 197, SEQ ID NO: 201, SEQID NO: 203, SEQ ID NO: 207, SEQ ID NO: 211, SEQ ID NO: 215, SEQ ID NO:219, SEQ ID NO: 223, SEQ ID NO: 227, SEQ ID NO: 231, SEQ ID NO: 235, SEQID NO: 239, SEQ ID NO: 243, SEQ ID NO: 247, SEQ ID NO: 251, SEQ ID NO:255, SEQ ID NO: 259, SEQ ID NO: 263, SEQ ID NO: 267, SEQ ID NO: 271, SEQID NO: 275, SEQ ID NO: 279, SEQ ID NO: 283, SEQ ID NO: 287, SEQ ID NO:291, SEQ ID NO: 295, SEQ ID NO: 299, SEQ ID NO: 303, SEQ ID NO: 307, andSEQ ID NO: 311; and a light chain variable region comprising an aminoacid sequence selected from the group of: SEQ ID NO: 193, SEQ ID NO:195, SEQ ID NO: 199, SEQ ID NO: 205, SEQ ID NO: 209, SEQ ID NO: 213, SEQID NO: 217, SEQ ID NO: 221, SEQ ID NO: 225, SEQ ID NO: 229, SEQ ID NO:233, SEQ ID NO: 237, SEQ ID NO: 241, SEQ ID NO: 245, SEQ ID NO: 249, SEQID NO: 253, SEQ ID NO: 257, SEQ ID NO: 261, SEQ ID NO: 265, SEQ ID NO:269, SEQ ID NO: 273, SEQ ID NO: 277, SEQ ID NO: 281, SEQ ID NO: 285, SEQID NO: 289, SEQ ID NO: 293, SEQ ID NO: 297, SEQ ID NO: 301, SEQ ID NO:305, and SEQ ID NO: 309. In one embodiment, the antibody comprises atleast one heavy or light chain variable region which does not comprisean amino acid sequence selected from the group of SEQ ID NO: 193, SEQ IDNO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203,SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ IDNO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO: 219, SEQ ID NO: 221,SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ IDNO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239,SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ IDNO: 249, SEQ ID NO: 251, SEQ ID NO: 253, and SEQ ID NO: 255.

In one embodiment, the antibody comprises an Fc region, particularly anIgG Fc region. In a further embodiment, the antibody is a full-lengthantibody, particularly an IgG class antibody. In another embodiment, theantibody comprises a human antibody constant region. In one embodiment,the antibody is human. In one embodiment, the antibody isglycoengineered to have modified oligosaccharides in the Fc region. Inone embodiment the antibody has an increased proportion ofnon-fucosylated and/or bisected oligosaccharides in the Fc region, ascompared to a non-glycoengineered antibody. In a further embodiment, theantibody has increased effector function and/or increased Fc receptorbinding affinity. In a particular embodiment, the increased effectorfunction is increased antibody-dependent cell-mediated cytotoxicity(ADCC). In another embodiment the antibody binds to FAP with a K_(D)value of lower than about 1 μM, preferably lower than about 100 nM, mostpreferably lower than about 1 nM or even lower than about 0.1 nM. In oneembodiment, the antibody is affinity matured. In one embodiment, theantibody binds to FAP in human tissues. In one embodiment, the antibodydoes not induce internalization of FAP.

In other aspects, the invention is also directed to polypeptides,polynucleotides, host cells, and expression vectors related to theantibodies. In a further aspect, the invention relates to methods ofmaking the antibodies. In a further aspect, the invention is directed tomethods of using the antibodies, particularly for the treatment ofdiseases characterized by expression of FAP, such as cancer.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, 1D, 1E, and 1F show Surface Plasmon Resonance(SPR)-based kinetic analyses of affinity-matured anti-FAP Fab fragments.Processed kinetic data sets are presented for clone 19G1 binding tohuman (hu) FAP (FIG. 1A) and murine (mu) FAP (FIG. 1B), for clone 20G8binding to hu FAP (FIG. 1C), mu FAP (FIG. 1D) and for clone 4B9 bindingto hu FAP (FIG. 1E) and mu FAP (FIG. 1F). Smooth lines represent aglobal fit of the data to a 1:1 interaction model.

FIGS. 2A, 2B, 2C, 2D, 2E, and 2F show SPR-based kinetic analyses ofaffinity-matured anti-FAP Fab fragments. Processed kinetic data sets arepresented for clone 5B8 binding to hu FAP (FIG. 2A) and mu FAP (FIG.2B), for clone 5F1 binding to hu FAP (FIG. 2C), mu FAP (FIG. 2D) and forclone 14B3 binding to hu FAP (FIG. 2E) and mu FAP (FIG. 2F). Smoothlines represent a global fit of the data to a 1:1 interaction model.

FIGS. 3A, 3B, 3C, 3D, 3E, and 3F show SPR-based kinetic analyses ofaffinity-matured anti-FAP Fab fragments. Processed kinetic data sets arepresented for clone 16F1 binding to hu FAP (FIG. 3A) and mu FAP (FIG.3B), for clone 16F8 binding to hu FAP (FIG. 3C), mu FAP (FIG. 3D) andfor clone O3C9 binding to hu FAP (FIG. 3E) and mu FAP (FIG. 3F). Smoothlines represent a global fit of the data to a 1:1 interaction model.

FIGS. 4A, 4B, 4C, 4D, 4E, 4F, 4G, and 4H show SPR-based kinetic analysesof affinity-matured anti-FAP Fab fragments. Processed kinetic data setsare presented for clone O2D7 binding to hu FAP (FIG. 4A) and mu FAP(FIG. 4B), for clone 28H1 binding to hu FAP (FIG. 4C), mu FAP (FIG. 4D),cyno FAP (FIG. 4E) and for clone 22A3 binding to hu FAP (FIG. 4F), muFAP (FIG. 4G) and Cynomolgus (cyno) FAP (FIG. 4H). Smooth linesrepresent a global fit of the data to a 1:1 interaction model.

FIGS. 5A, 5B, 5C, 5D, 5E, and 5F show SPR-based kinetic analyses ofaffinity-matured anti-FAP Fab fragments. Processed kinetic data sets arepresented for clone 29B11 binding to hu FAP (FIG. 5A), mu FAP (FIG. 5B),cyno FAP (FIG. 5C) and for clone 23C10 binding to hu FAP (FIG. 5D), muFAP (FIG. 5E) and cyno FAP (FIG. 5F). Smooth lines represent a globalfit of the data to a 1:1 interaction model.

FIGS. 6A1, 6A2, 6A3, 6B1, 6B2, 6B3, 6C1, 6C2, and 6C3 show SPR-basedkinetic analyses of 3F2 (FIG. 6A1, 6A2, 6A3), 4G8 (FIG. 6B1, 6B2, 6B3)and 3D9 (FIG. 6C1, 6C2, 6C3) anti-FAP antibodies binding as Fabfragments to human, mouse and cynomolgus FAP. Processed kinetic datasets are presented, smooth lines represent a global fit of the data to a1:1 interaction model.

FIGS. 7A1, 7A2, 7A3, 7B1, 7B2, 7B3, 7C1, 7C2, and 7C3 show SPR-basedkinetic analyses of 3F2 (FIG. 7A1, 7A2, 7A3), 4G8 (FIG. 7B1, 7B2, 7B3)and 3D9 (FIG. 7C1, 7C2, 7C3) anti-FAP antibodies binding as human IgG tohuman, mouse and cynomolgus FAP. Processed kinetic data sets arepresented, smooth lines represent a global fit of the data to a 1:1interaction model.

FIGS. 8A1, 8A2, 8B1, 8B2, 8C1, 8C2, 8D1 and 8D2 show a representativepictures of human samples of (FIG. 8A1) non-small cell lung cancer(NSCLC) immunohistochemically stained for FAP using 2F3 mouse IgG2aantibody, (FIG. 8B1) colon adenocarcinoma immunohistochemically stainedfor FAP using 2F3 mouse IgG2a antibody, (FIG. 8C1) colon adenocarcinomaimmunohistochemically stained for FAP using 3D9 mouse IgG2a antibody,and (FIG. 8D1) colon adenocarcinoma immunohistochemically stained forFAP using 4G8 mouse IgG2a antibody. FAP is detected in the tumor stromain all samples and by all antibodies (left panels (FIG. 8A1, FIG. 8B1,FIG. 8C1, FIG. 8D1), while no staining is observed for the isotypecontrol antibody (right panels (FIG. 8A2, FIG. 8B2, FIG. 8C2, FIG.8D2)).

FIGS. 9A and 9B show binding of human IgG1 anti-FAP antibodies to FAPexpressed on HEK 293 cells stably transfected with human (FIG. 9A) ormurine (FIG. 9B) FAP, as determined by FACS.

FIG. 10 shows binding of human IgG1 anti-FAP antibodies to DPPIV (CD26)or HER2 expressed on stably transfected HEK 293 cells, as determined byFACS. Anti-HER2 antibody trastuzumab and anti-CD26 antibody were used aspositive controls. Secondary antibody, control IgG or no antibody at all(cells only) were used as negative controls.

FIGS. 11A, 11B, 11C and 11D show binding of human IgG1 anti-FAPantibodies to FAP on human fibroblasts (cell line GM05389), asdetermined by FACS. Secondary antibody or no antibody at all were usedas negative controls.

FIG. 12 shows binding of human IgG1 anti-FAP antibodies to humanfibroblasts (cell line GM05389), different human tumor cell lines, orHEK 293 cells stably transfected with human FAP, as determined by FACS.

FIGS. 13A and 13B show expression levels of FAP on the surface ofGM05389 lung fibroblasts at different time points after incubation withthe anti-FAP human IgG1 antibodies 3F2 or 4G8, as determined by FACS. Nosignificant decrease in FAP expression levels, indicatinginternalization of FAP, was observed. Secondary antibody alone is shownas negative control.

FIGS. 14A1, 14A2, 14A3, 14A4, 14B1, 14B2, 14B3, 14B4, 14C1, 14C2, 14C3,14C4, 14D1, 14D2, 14D3, and 14D4 present representativeimmunofluorescence images showing plasma membrane staining on GM05389lung fibroblasts obtained after binding of anti-FAP 4G8 IgG for 45 minat 4° C. (FIG. 14A1), for 20 min at 37° C. (FIG. 14B1), for 1 hour at37° C. (FIG. 14C1) or for 6 hours at 37° C. (FIG. 14D1). The anti-CD20antibody GA101, used as isotype control, shows background staining (FIG.14A3, FIG. 14B3, FIG. 14C3, FIG. 14D3). EEA1 labels early endosomes(FIG. 14A2, FIG. 14B2, FIG. 14C2, FIG. 14D2; GA101 isotype control isshown in FIG. 14A4, FIG. 14B4, FIG. 14C4, FIG. 14D4)). Note thepersistence of the FAP surface plasma membrane staining up to 6 hoursafter anti-FAP 4G8 antibody binding.

FIGS. 15A, 15B, 15C, and 15D show the purification and analysis of thewild-type 28H1 human IgG. FIG. 15A) Protein A affinity chromatographypurification step. FIG. 15B) Size exclusion chromatography purificationstep. FIG. 15C) Analytical SDS PAGE. FIG. 15D) Analytical size exclusionchromatography. Experimental procedures are described in Example 1.

FIGS. 16A, 16B, 16C, and 16D show the purification and analysis of theglycoengineered 28H1 human IgG. FIG. 16A) Protein A affinitychromatography purification step. FIG. 16B) Size exclusionchromatography purification step. FIG. 16C) Analytical SDS PAGE. FIG.16D) Analytical size exclusion chromatography. Experimental proceduresare described in Example 1.

FIGS. 17A, 17B, 17C, and 17D show the purification and analysis of thewild-type 29B11 human IgG. FIG. 17A) Protein A affinity chromatographypurification step. FIG. 17B) Size exclusion chromatography purificationstep. FIG. 17C) Analytical SDS PAGE. FIG. 17D) Analytical size exclusionchromatography. Experimental procedures are described in Example 1.

FIGS. 18A, 18B, 18C, and 18D show the purification and analysis of theglycoengineered 29B11 human IgG. FIG. 18A) Protein A affinitychromatography purification step. FIG. 18B) Size exclusionchromatography purification step. FIG. 18C) Analytical SDS PAGE. FIG.18D) Analytical size exclusion chromatography. Experimental proceduresare described in Example 1.

FIGS. 19A, 19B, 19C, and 19D show the purification and analysis of thewild-type 3F2 human IgG. FIG. 19A) Protein A affinity chromatographypurification step. FIG. 19B) Size exclusion chromatography purificationstep. FIG. 19C) Analytical SDS PAGE. FIG. 19D) Analytical size exclusionchromatography. Experimental procedures are described in Example 1.

FIGS. 20A, 20B, 20C, and 20D show the purification and analysis of theglycoengineered 3F2 human IgG. FIG. 20A) Protein A affinitychromatography purification step. FIG. 20B) Size exclusionchromatography purification step. FIG. 20C) Analytical SDS PAGE. FIG.20D) Analytical size exclusion chromatography. Experimental proceduresare described in Example 1.

FIGS. 21A, 21B, 21C, and 21D show the purification and analysis of thewild-type 4G8 human IgG. FIG. 21A) Protein A affinity chromatographypurification step. FIG. 21B) Size exclusion chromatography purificationstep. FIG. 21C) Analytical SDS PAGE. FIG. 21D) Analytical size exclusionchromatography. Experimental procedures are described in Example 1.

FIGS. 22A, 22B, 22C, and 22D show the purification and analysis of theglycoengineered 4G8 human IgG. FIG. 22A) Protein A affinitychromatography purification step. FIG. 22B) Size exclusionchromatography purification step. FIG. 22C) Analytical SDS PAGE. FIG.22D) Analytical size exclusion chromatography. Experimental proceduresare described in Example 1.

FIG. 23 shows binding of affinity matured anti-FAP antibody 28H1 tohuman FAP on HEK293 cells compared to binding of the parental 4G8anti-FAP antibody.

FIG. 24 shows the results of an LDH release assay to test ADCC mediatedby the anti-FAP IgG antibodies 28H1 (affinity matured) and 4G8, 3F8(parentals) as wildtype (wt) and glycoengineered (ge) versions, withHEK293-hFAP as target cells and PBMNCs as effector cells (F/F FcγRIIIagenotype).

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION I. DEFINITIONS

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (K_(D)), which is the ratio of dissociation and associationrate constants (k_(off) and k_(on), respectively). Thus, equivalentaffinities may comprise different rate constants, as long as the ratioof the rate constants remains the same. Affinity can be measured bycommon methods known in the art, including those described herein.Specific illustrative and exemplary embodiments for measuring bindingaffinity are described in the following.

An “affinity matured” antibody refers to an antibody with one or morealterations (e.g. amino acid mutations) in one or more hypervariableregions (HVRs) (e.g. CDRs), compared to a parent antibody which does notpossess such alterations, such alterations resulting in an improvementin the affinity of the antibody for antigen. Typically, the affinitymatured antibody binds to the same epitope as the parent antibody.

The terms “anti-FAP antibody” and “an antibody that binds to FibroblastActivation Protein (FAP)” refer to an antibody that is capable ofbinding FAP with sufficient affinity such that the antibody is useful asa diagnostic and/or therapeutic agent in targeting FAP. In oneembodiment, the extent of binding of an anti-FAP antibody to anunrelated, non-FAP protein is less than about 10% of the binding of theantibody to FAP as measured, e.g., by a radioimmunoassay (RIA) or flowcytometry (FACS). In one embodiment, the extent of binding of ananti-FAP antibody of the invention to DPPIV, a protein closely relatedto FAP (also known as CD26; GenBank Accession Number P27487), is lessthan about 15%, about 10% or about 5% of the binding of the antibody toFAP as measured by FACS. In certain embodiments, an antibody that bindsto FAP has a dissociation constant (K_(D)) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸Mto 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³ M). In certain embodiments, ananti-FAP antibody binds to an epitope of FAP that is conserved among FAPfrom different species.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity. Also included are antibodyfragments having an Fc region, and fusion proteins that comprise aregion equivalent to the Fc region of an immunoglobulin.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂, single-chain antibody molecules (e.g. scFv), diabodies, andmultispecific antibodies formed from antibody fragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “antigen binding domain” refers to the part of an antigenbinding molecule that comprises the area which specifically binds to andis complementary to part or all of an antigen. Where an antigen islarge, an antigen binding molecule may only bind to a particular part ofthe antigen, which part is termed an epitope. An antigen binding domainmay be provided by, for example, one or more antibody variable domains(also called antibody variable regions). Preferably, an antigen bindingdomain comprises an antibody light chain variable region (VL) and anantibody heavy chain variable region (VH).

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species. For chimeric antibodies, forexample, the non-antigen binding components may be derived from a widevariety of species, including primates such as chimpanzees and humans.Humanized antibodies are a particularly preferred form of chimericantibodies.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, and IgA₂. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents a cellular function and/or causes cell death ordestruction. Cytotoxic agents include, but are not limited to,radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³,Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeuticagents or drugs (e.g., methotrexate, adriamicin, vinca alkaloids(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycinC, chlorambucil, daunorubicin or other intercalating agents); growthinhibitory agents; enzymes and fragments thereof such as nucleolyticenzymes; antibiotics; toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof; and the variousantitumor or anticancer agents disclosed below.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis;cytokine secretion; immune-complex-mediated antigen uptake by antigenpresenting cells; down regulation of cell surface receptors (e.g. B cellreceptor); and B cell activation.

An “effective amount” of an agent, e.g., a pharmaceutical formulation,refers to an amount effective, at dosages and for periods of timenecessary, to achieve the desired therapeutic or prophylactic result.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

A “region equivalent to the Fc region of an immunoglobulin” is intendedto include naturally occurring allelic variants of the Fc region of animmunoglobulin as well as variants having alterations which producesubstitutions, additions, or deletions but which do not decreasesubstantially the ability of the immunoglobulin to mediate effectorfunctions (such as antibody-dependent cellular cytotoxicity). Forexample, one or more amino acids can be deleted from the N-terminus orC-terminus of the Fc region of an immunoglobulin without substantialloss of biological function. Such variants can be selected according togeneral rules known in the art so as to have minimal effect on activity(see, e.g., Bowie, J. U. et al., Science 247:1306-10 (1990)).

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) (or CDR) residues. The FR of a variabledomain generally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.In one embodiment, the host cell is engineered to allow the productionof an antibody with modified oligosaccharides. In certain embodiments,the host cells have been further manipulated to express increased levelsof one or more polypeptides havingβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII) activity. Host cellsinclude cultured cells, e.g., mammalian cultured cells, such as CHOcells, BHK cells, NS0 cells, SP2/0 cells, YO myeloma cells, P3X63 mousemyeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells,insect cells, and plant cells, to name only a few, but also cellscomprised within a transgenic animal, transgenic plant or cultured plantor animal tissue. A “human antibody” is one which possesses an aminoacid sequence which corresponds to that of an antibody produced by ahuman or a human cell or derived from a non-human source that utilizeshuman antibody repertoires or other human antibody-encoding sequences.This definition of a human antibody specifically excludes a humanizedantibody comprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR”, as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition.With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. Hypervariable regions (HVRs)are also referred to as complementarity determining regions (CDRs), andthese terms are used herein interchangeably in reference to portions ofthe variable region that form the antigen binding regions. Thisparticular region has been described by Kabat et al., U.S. Dept. ofHealth and Human Services, “Sequences of Proteins of ImmunologicalInterest” (1983) and by Chothia et al., J. Mol. Biol. 196:901-917(1987), where the definitions include overlapping or subsets of aminoacid residues when compared against each other. Nevertheless,application of either definition to refer to a CDR of an antibody orvariants thereof is intended to be within the scope of the term asdefined and used herein. The appropriate amino acid residues whichencompass the CDRs as defined by each of the above cited references areset forth below in Table 1 as a comparison. The exact residue numberswhich encompass a particular CDR will vary depending on the sequence andsize of the CDR. Those skilled in the art can routinely determine whichresidues comprise a particular CDR given the variable region amino acidsequence of the antibody.

TABLE 1 CDR Definitions¹ CDR Kabat Chothia AbM² V_(H) CDR1 31-35 26-3226-35 V_(H) CDR2 50-65 52-58 50-58 V_(H) CDR3  95-102  95-102  95-102V_(L) CDR1 24-34 26-32 24-34 V_(L) CDR2 50-56 50-52 50-56 V_(L) CDR389-97 91-96 89-97 ¹Numbering of all CDR definitions in Table 1 isaccording to the numbering conventions set forth by Kabat et al. (seebelow). ²“AbM” with a lowercase “b” as used in Table 1 refers to theCDRs as defined by Oxford Molecular's “AbM” antibody modeling software.

Kabat et al. also defined a numbering system for variable regionsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable region sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).Unless otherwise specified, references to the numbering of specificamino acid residue positions in an antibody variable region areaccording to the Kabat numbering system.

CDRs also comprise “specificity determining residues,” or “SDRs,” whichare residues that contact antigen. SDRs are contained within regions ofthe CDRs called abbreviated-CDRs, or a-CDRs. In general, only one-fifthto one-third of the residues in a given CDR participate in antigenbinding. The specificity-determining residues in a particular CDR can beidentified by, for example, computation of interatomic contacts fromthree-dimensional modeling and determination of the sequence variabilityat a given residue position in accordance with the methods described inPadlan et al., FASEB J. 9(1):133-139 (1995). Exemplary a-CDRs (a-CDR-L1,a-CDR-L2, a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at aminoacid residues 31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58of H2, and 95-102 of H3 (see Almagro and Fransson, Front. Biosci.13:1619-1633 (2008).)

An “antibody conjugate” is an antibody conjugated to a cytotoxic agent.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC) methods. For review of methods for assessment of antibody purity,see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” polynucleotide refers to a polynucleotide molecule thathas been separated from a component of its natural environment. Anisolated polynucleotide includes a polynucleotide molecule contained incells that ordinarily contain the polynucleotide molecule, but thepolynucleotide molecule is present extrachromosomally or at achromosomal location that is different from its natural chromosomallocation.

“Isolated polynucleotide encoding an anti-FAP antibody” refers to one ormore polynucleotide molecules encoding antibody heavy and light chains(or fragments thereof), including such polynucleotide molecule(s) in asingle vector or separate vectors, and such polynucleotide molecule(s)present at one or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3),also called a heavy chain constant region. Similarly, from N- toC-terminus, each light chain has a variable region (VL), also called avariable light domain or a light chain variable domain, followed by aconstant light (CL) domain, also called a light chain constant region.The light chain of an antibody may be assigned to one of two types,called kappa (κ) and lambda (λ), based on the amino acid sequence of itsconstant domain.

“No substantial cross-reactivity” means that a molecule (e.g., anantibody) does not recognize or specifically bind an antigen differentfrom the actual target antigen of the molecule (e.g. an antigen closelyrelated to the target antigen), particularly when compared to thattarget antigen. For example, an antibody may bind less than about 10% toless than about 5% to an antigen different from the actual targetantigen, or may bind said antigen different from the actual targetantigen at an amount selected from the group consisting of less thanabout 10%, 9%, 8% 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.2%, or 0.1%,preferably less than about 2%, 1%, or 0.5%, and most preferably lessthan about 0.2% or 0.1% antigen different from the actual targetantigen.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

The term “parent” antibody refers to an antibody that is used as thestarting point or basis for the preparation of a variant.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A. Unless specifically stated otherwise, all % aminoacid sequence identity values used herein are obtained as described inthe immediately preceding paragraph using the ALIGN-2 computer program.

Similarly, by a nucleic acid or polynucleotide having a nucleotidesequence at least, for example, 95% “identical” to a referencenucleotide sequence of the present invention, it is intended that thenucleotide sequence of the polynucleotide is identical to the referencesequence except that the polynucleotide sequence may include up to fivepoint mutations per each 100 nucleotides of the reference nucleotidesequence. In other words, to obtain a polynucleotide having a nucleotidesequence at least 95% identical to a reference nucleotide sequence, upto 5% of the nucleotides in the reference sequence may be deleted orsubstituted with another nucleotide, or a number of nucleotides up to 5%of the total nucleotides in the reference sequence may be inserted intothe reference sequence. These alterations of the reference sequence mayoccur at the 5′ or 3′ terminal positions of the reference nucleotidesequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence. As a practicalmatter, whether any particular polynucleotide or polypeptide is at least80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a nucleotidesequence or polypeptide sequence of the present invention can bedetermined conventionally using known computer programs, such as theones listed above. The term “pharmaceutical formulation” refers to apreparation which is in such form as to permit the biological activityof an active ingredient contained therein to be effective, and whichcontains no additional components which are unacceptably toxic to asubject to which the formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

The term “Fibroblast Activation Protein (FAP)” as used herein, refers toany native FAP from any vertebrate source, including mammals such asprimates (e.g. humans, see GenBank Accession Number AAC51668) androdents (e.g., mice, see GenBank Accession Number AAH19190), unlessotherwise indicated. The term encompasses “full-length,” unprocessed FAPas well as any form of FAP that results from processing in the cell. Theterm also encompasses naturally occurring variants of FAP, e.g., splicevariants or allelic variants. Preferably, an anti-FAP antibody of theinvention binds to the extracellular domain of FAP. The amino acidsequence of exemplary human, mouse and cynomolgus monkey FAP ectodomains(with a C-terminal poly-lysine and 6× His-tag) are shown in SEQ ID NO:317, SEQ ID NO: 319, and SEQ ID NO: 321 respectively.

As used herein, “treatment” (and grammatical variations thereof such as“treat” or “treating”) refers to clinical intervention in an attempt toalter the natural course of disease of the individual being treated, andcan be performed either for prophylaxis or during the course of clinicalpathology. Desirable effects of treatment include, but are not limitedto, preventing occurrence or recurrence of disease, alleviation ofsymptoms, diminishment of any direct or indirect pathologicalconsequences of the disease, preventing metastasis, decreasing the rateof disease progression, amelioration or palliation of the disease state,and remission or improved prognosis. In some embodiments, antibodies ofthe invention are used to delay development of a disease or to slow theprogression of a disease.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6^(th) ed., W.H. Freeman and Co., page 91(2007).) A single VH or VL domain may be sufficient to conferantigen-binding specificity. Furthermore, antibodies that bind aparticular antigen may be isolated using a VH or VL domain from anantibody that binds the antigen to screen a library of complementary VLor VH domains, respectively. See, e.g., Portolano et al., J. Immunol.150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

As used herein, the term “polypeptide having GnTIII activity” refers topolypeptides that are able to catalyze the addition of aN-acetylglucosamine (GlcNAc) residue in β-1-4 linkage to the β-linkedmannoside of the trimannosyl core of N-linked oligosaccharides. Thisincludes fusion polypeptides exhibiting enzymatic activity similar to,but not necessarily identical to, an activity ofβ(1,4)-N-acetylglucosaminyltransferase III, also known asβ-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyl-transferase (EC2.4.1.144), according to the Nomenclature Committee of the InternationalUnion of Biochemistry and Molecular Biology (NC-IUBMB), as measured in aparticular biological assay, with or without dose dependency. In thecase where dose dependency does exist, it need not be identical to thatof GnTIII, but rather substantially similar to the dose-dependence in agiven activity as compared to the GnTIII (i.e., the candidatepolypeptide will exhibit greater activity or not more than about 25-foldless and, preferably, not more than about tenfold less activity, andmost preferably, not more than about three-fold less activity relativeto the GnTIII).

As used herein, the term “Golgi localization domain” refers to the aminoacid sequence of a Golgi resident polypeptide which is responsible foranchoring the polypeptide to a location within the Golgi complex.Generally, localization domains comprise amino terminal “tails” of anenzyme.

As used herein, the terms “engineer, engineered, engineering,”particularly with the prefix “glyco-,” as well as the term“glycosylation engineering” are considered to include any manipulationof the glycosylation pattern of a naturally occurring or recombinantpolypeptide or fragment thereof. Glycosylation engineering includesmetabolic engineering of the glycosylation machinery of a cell,including genetic manipulations of the oligosaccharide synthesispathways to achieve altered glycosylation of glycoproteins expressed incells. Furthermore, glycosylation engineering includes the effects ofmutations and cell environment on glycosylation. In one embodiment, theglycosylation engineering is an alteration in glycosyltransferaseactivity. In a particular embodiment, the engineering results in alteredglucosaminyltransferase activity and/or fucosyltransferase activity.

As used herein, the term “Fc-mediated cellular cytotoxicity” includesantibody-dependent cell-mediated cytotoxicity (ADCC) and cellularcytotoxicity mediated by a soluble Fc-fusion protein containing a humanFc-region. It is an immune mechanism leading to the lysis of “targetedcells” by “human immune effector cells.”

As used herein, the term “human immune effector cells” refers to apopulation of leukocytes that display Fc receptors on their surfaces,through which they bind to the Fc-region of antibodies or of Fc-fusionproteins and perform effector functions. Such a population may include,but is not limited to, peripheral blood mononuclear cells (PBMC) and/ornatural killer (NK) cells.

As used herein, the term “targeted cells” refers to cells to whichantigen binding molecules comprising an Fc region (e.g., antibodies orfragments thereof comprising an Fc region) or Fc-fusion proteinsspecifically bind. The antigen binding molecules or Fc fusion-proteinsbind to target cells via the protein part that is N-terminal to the Fcregion.

As used herein, the term “increased Fc-mediated cellular cytotoxicity”is defined as either an increase in the number of “targeted cells” thatare lysed in a given time, at a given concentration of antibody or ofFc-fusion protein in the medium surrounding the target cells, by themechanism of Fc-mediated cellular cytotoxicity defined above, and/or areduction in the concentration of antibody or of Fc-fusion protein, inthe medium surrounding the target cells, required to achieve the lysisof a given number of “targeted cells,” in a given time, by the mechanismof Fc-mediated cellular cytotoxicity. The increase in Fc-mediatedcellular cytotoxicity is relative to the cellular cytotoxicity mediatedby the same antigen binding molecule or Fc-fusion protein produced bythe same type of host cells, using the same standard production,purification, formulation and storage methods, (which are known to thoseskilled in the art) but that has not been produced by host cellsengineered to have an altered pattern of glycosylation (e.g., to expressthe glycosyltransferase, GnTIII, or other glycosyltransferases) by themethods described herein.

By “antibody having increased antibody dependent cell-mediatedcytotoxicity (ADCC)” is meant an antibody, as that term is definedherein, having increased ADCC as determined by any suitable method knownto those of ordinary skill in the art. One accepted in vitro ADCC assayis as follows:

-   -   1) the assay uses target cells that are known to express the        target antigen recognized by the antigen-binding region of the        antibody;    -   2) the assay uses human peripheral blood mononuclear cells        (PBMCs), isolated from blood of a randomly chosen healthy donor,        as effector cells;    -   3) the assay is carried out according to following protocol:    -   i) the PBMCs are isolated using standard density centrifugation        procedures and are suspended at 5×10⁶ cells/ml in RPMI cell        culture medium;    -   ii) the target cells are grown by standard tissue culture        methods, harvested from the exponential growth phase with a        viability higher than 90%, washed in RPMI cell culture medium,        labeled with 100 micro-Curies of ⁵¹Cr, washed twice with cell        culture medium, and resuspended in cell culture medium at a        density of 10⁵ cells/ml;    -   iii) 100 microliters of the final target cell suspension above        are transferred to each well of a 96-well microtiter plate;    -   iv) the antibody is serially-diluted from 4000 ng/ml to 0.04        ng/ml in cell culture medium and 50 microliters of the resulting        antibody solutions are added to the target cells in the 96-well        microtiter plate, testing in triplicate various antibody        concentrations covering the whole concentration range above;    -   v) for the maximum release (MR) controls, 3 additional wells in        the plate containing the labeled target cells, receive 50        microliters of a 2% (V/V) aqueous solution of non-ionic        detergent (Nonidet, Sigma, St. Louis), instead of the antibody        solution (point iv above);    -   vi) for the spontaneous release (SR) controls, 3 additional        wells in the plate containing the labeled target cells, receive        50 microliters of RPMI cell culture medium instead of the        antibody solution (point iv above);    -   vii) the 96-well microtiter plate is then centrifuged at 50×g        for 1 minute and incubated for 1 hour at 4° C.;    -   viii) 50 microliters of the PBMC suspension (point i above) are        added to each well to yield an effector:target cell ratio of        25:1 and the plates are placed in an incubator under 5% CO₂        atmosphere at 37° C. for 4 hours;    -   ix) the cell-free supernatant from each well is harvested and        the experimentally released radioactivity (ER) is quantified        using a gamma counter;    -   x) the percentage of specific lysis is calculated for each        antibody concentration according to the formula        (ER−MR)/(MR−SR)×100, where ER is the average radioactivity        quantified (see point ix above) for that antibody concentration,        MR is the average radioactivity quantified (see point ix above)        for the MR controls (see point v above), and SR is the average        radioactivity quantified (see point ix above) for the SR        controls (see point vi above);    -   4) “increased ADCC” is defined as either an increase in the        maximum percentage of specific lysis observed within the        antibody concentration range tested above, and/or a reduction in        the concentration of antibody required to achieve one half of        the maximum percentage of specific lysis observed within the        antibody concentration range tested above. The increase in ADCC        is relative to the ADCC, measured with the above assay, mediated        by the same antibody, produced by the same type of host cells,        using the same standard production, purification, formulation        and storage methods, which are known to those skilled in the        art, but that has not been produced by host cells engineered to        overexpress GnTIII.

II. COMPOSITIONS AND METHODS

Fibroblast Activation Protein (FAP) is expressed in the majority oftumors but essentially absent from healthy adult tissues, thusantibodies targeting this antigen have great therapeutic potential. Thepresent invention provides antibodies that bind to FAP, in particularantibodies with high affinity and strong effector functions. Antibodiesof the invention are useful, e.g., for the diagnosis or treatment ofdiseases characterized by expression of FAP, such as cancer.

A. Exemplary Anti-FAP Antibodies

The present invention provides for antibodies that specifically bind toFibroblast Activation Protein (FAP). Particularly, the present inventionprovides for antibodies that specifically bind FAP, wherein saidantibodies are glycoengineered to have increased effector function.

In one embodiment, an anti-FAP antibody of the invention comprises atleast one (e.g. one, two, three, four, five, or six) heavy or lightchain complementarity determining region (CDR) selected from the groupof SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ IDNO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO:49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ IDNO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO:87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ IDNO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105,SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ IDNO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123,SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ IDNO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141,SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ IDNO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159,SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ IDNO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, and SEQ ID NO:177, or a variant or truncated form thereof containing at least thespecificity-determining residues (SDRs) for said CDR.

In one embodiment, said at least one CDR is a heavy chain CDR,particularly a heavy chain CDR3 selected from the group of SEQ ID NO:135, SEQ ID NO: 137, SEQ ID NO: 139, and SEQ ID NO: 141. In anotherembodiment, the antibody comprises at least one heavy chain CDR and atleast one light chain CDR, particularly a heavy chain CDR3 selected fromthe group of SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, and SEQ IDNO: 141, and a light chain CDR3 selected from the group of SEQ ID NO:163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQID NO: 173, SEQ ID NO: 175 and SEQ ID NO: 177.

In one embodiment, an antibody of the invention comprises at least one,at least two, or all three heavy chain CDR (HCDR) sequences selectedfrom (a) HCDR1 comprising an amino acid sequence selected from the groupof SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO:11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ IDNO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQID NO: 31, and SEQ ID NO: 33; (b) HCDR2 comprising an amino acidsequence selected from the group of SEQ ID NO: 35, SEQ ID NO: 37, SEQ IDNO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57,SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO:67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ IDNO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95,SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO:105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO:123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, andSEQ ID NO: 133; and (c) HCDR3 comprising an amino acid sequence selectedfrom the group of SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, andSEQ ID NO: 141.

In a further embodiment, the antibody comprises a heavy chain variableregion comprising (a) a heavy chain CDR1 selected from the group of SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO:21,SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO:31, and SEQ ID NO: 33; (b) a heavy chain CDR2 selected from the group ofSEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO:43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ IDNO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71,SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO:81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ IDNO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO:109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO:127, SEQ ID NO: 129, SEQ ID NO: 131, and SEQ ID NO: 133; and (c) a heavychain CDR3 selected from the group of SEQ ID NO: 135, SEQ ID NO: 137,SEQ ID NO: 139, and SEQ ID NO: 141, or variants or truncated formsthereof containing at least the SDRs for said CDRs.

In one embodiment, an antibody of the invention comprises at least one,at least two, or all three light chain CDR (LCDR) sequences selectedfrom (a) LCDR1 comprising an amino acid sequence selected from the groupof SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, and SEQ ID NO: 149;(b) LCDR2 comprising an amino acid sequence selected from the group ofSEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ IDNO: 159, and SEQ ID NO: 161; and (c) LCDR3 comprising an amino acidsequence selected from the group of SEQ ID NO: 163, SEQ ID NO: 165, SEQID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO:175, and SEQ ID NO: 177. In a further embodiment, the antibody comprisesa light chain variable region comprising (a) a light chain CDR1 selectedfrom the group of SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, andSEQ ID NO: 149; (b) a light chain CDR2 selected from the group of SEQ IDNO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159,and SEQ ID NO: 161; and (c) a light chain CDR3 selected from the groupof SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, and SEQ ID NO: 177, orvariants or truncated forms thereof containing at least the SDRs forsaid CDRs.

In a more specific embodiment, an antibody of the invention comprises aheavy chain variable region comprising a heavy chain CDR1 selected fromthe group of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19,SEQ ID NO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO:29, SEQ ID NO: 31, and SEQ ID NO: 33; a heavy chain CDR2 selected fromthe group of SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41,SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO:51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ IDNO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79,SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO:89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ IDNO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107,SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ IDNO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125,SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, and SEQ ID NO: 133; anda heavy chain CDR3 selected from the group of SEQ ID NO: 135, SEQ ID NO:137, SEQ ID NO: 139, and SEQ ID NO: 141, and a light chain variableregion comprising a light chain CDR1 selected from the group of SEQ IDNO: 143, SEQ ID NO: 145, SEQ ID NO: 147, and SEQ ID NO: 149; a lightchain CDR2 selected from the group of SEQ ID NO: 151, SEQ ID NO: 153,SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, and SEQ ID NO: 161; anda light chain CDR3 selected from the group of SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQID NO: 175, and SEQ ID NO: 177, or variants or truncated forms thereofcontaining at least the SDRs for said CDRs.

In another embodiment, an antibody of the invention comprises a heavychain variable region comprising a heavy chain CDR1 selected from thegroup of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29,SEQ ID NO: 31, and SEQ ID NO: 33; a heavy chain CDR2 selected from thegroup of SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51,SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ IDNO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89,SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO:99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO:117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, and SEQ ID NO: 133; and aheavy chain CDR3 selected from the group of SEQ ID NO: 135, SEQ ID NO:137, SEQ ID NO: 139, and SEQ ID NO: 141, and a light chain variableregion comprising a light chain CDR1 selected from the group of SEQ IDNO: 143, SEQ ID NO: 145, SEQ ID NO: 147, and SEQ ID NO: 149; a lightchain CDR2 selected from the group of SEQ ID NO: 151, SEQ ID NO: 153,SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, and SEQ ID NO: 161; anda light chain CDR3 selected from the group of SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQID NO: 175, and SEQ ID NO: 177, wherein at least one of said CDRs isselected from the group of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11,SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 29, SEQ ID NO:31, SEQ ID NO: 33, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ IDNO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO:85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ IDNO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 109, SEQ ID NO: 111,SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ IDNO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129,SEQ ID NO: 131, SEQ ID NO: 133 and SEQ ID NO: 177.

In another embodiment, an antibody of the invention comprises a heavychain variable region comprising a heavy chain CDR1 selected from thegroup of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQID NO:21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29,SEQ ID NO: 31, and SEQ ID NO: 33; a heavy chain CDR2 selected from thegroup of SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51,SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO:61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ IDNO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89,SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO:99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO:117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, and SEQ ID NO: 133; and aheavy chain CDR3 selected from the group of SEQ ID NO: 135, SEQ ID NO:137, SEQ ID NO: 139, and SEQ ID NO: 141, and a light chain variableregion comprising a light chain CDR1 selected from the group of SEQ IDNO: 143, SEQ ID NO: 145, SEQ ID NO: 147, and SEQ ID NO: 149; a lightchain CDR2 selected from the group of SEQ ID NO: 151, SEQ ID NO: 153,SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, and SEQ ID NO: 161; anda light chain CDR3 selected from the group of SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQID NO: 175, and SEQ ID NO: 177, wherein at least one of said CDRs is nota CDR selected from the group of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ IDNO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 69, SEQID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 101, SEQ ID NO: 103,SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 135, SEQ ID NO: 137, SEQ IDNO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147,SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ IDNO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165,SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, and SEQID NO: 175.

In another embodiment, an antibody of the invention comprises a heavychain variable region comprising a heavy chain CDR1 selected from thegroup of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 15, SEQID NO: 23, SEQ ID NO: 25, and SEQ ID NO: 27; a heavy chain CDR2 selectedfrom the group of SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ IDNO: 41, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, and SEQ ID NO: 107; and aheavy chain CDR3 selected from the group of SEQ ID NO: 135, SEQ ID NO:137, SEQ ID NO: 139, and SEQ ID NO: 141, and a light chain variableregion comprising a light chain CDR1 selected from the group of SEQ IDNO: 143, SEQ ID NO: 145, SEQ ID NO: 147, and SEQ ID NO: 149; a lightchain CDR2 selected from the group of SEQ ID NO: 151, SEQ ID NO: 153,SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, and SEQ ID NO: 161; anda light chain CDR3 selected from the group of SEQ ID NO: 163, SEQ ID NO:165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, andSEQ ID NO: 175, or variants or truncated forms thereof containing atleast the SDRs for said CDRs.

In a specific embodiment, an antibody of the invention comprises a heavychain variable region comprising a heavy chain CDR1 selected from thegroup of SEQ ID NO: 3, SEQ ID NO: 13, and SEQ ID NO: 23; a heavy chainCDR2 selected from the group of SEQ ID NO: 35, SEQ ID NO: 69, and SEQ IDNO: 101; and the heavy chain CDR3 of SEQ ID NO: 135, and a light chainvariable region comprising the light chain CDR1 of SEQ ID NO: 143, thelight chain CDR2 of SEQ ID NO: 151, and the light chain CDR3 of SEQ IDNO: 163. In another specific embodiment, an antibody of the inventioncomprises a heavy chain variable region comprising a heavy chain CDR1selected from the group of SEQ ID NO: 3, SEQ ID NO: 13, and SEQ ID NO:23; a heavy chain CDR2 selected from the group of SEQ ID NO: 37, SEQ IDNO: 71, and SEQ ID NO: 103; and the heavy chain CDR3 of SEQ ID NO: 137,and a light chain variable region comprising the light chain CDR1 of SEQID NO: 145, the light chain CDR2 of SEQ ID NO: 153, and the light chainCDR3 of SEQ ID NO: 165. In yet another specific embodiment, an antibodyof the invention comprises a heavy chain variable region comprising aheavy chain CDR1 selected from the group of SEQ ID NO: 3, SEQ ID NO: 13,and SEQ ID NO: 23; a heavy chain CDR2 selected from the group of SEQ IDNO: 35, SEQ ID NO: 69, and SEQ ID NO: 101; and the heavy chain CDR3 ofSEQ ID NO: 137, and a light chain variable region comprising the lightchain CDR1 of SEQ ID NO: 147, the light chain CDR2 of SEQ ID NO: 155,and the light chain CDR3 of SEQ ID NO: 167. In another specificembodiment, an antibody of the invention comprises a heavy chainvariable region comprising a heavy chain CDR1 selected from the group ofSEQ ID NO: 3, SEQ ID NO: 13, and SEQ ID NO: 23; a heavy chain CDR2selected from the group of SEQ ID NO: 39, SEQ ID NO: 73, and SEQ ID NO:105; and the heavy chain CDR3 of SEQ ID NO: 135, and a light chainvariable region comprising the light chain CDR1 of SEQ ID NO: 145, thelight chain CDR2 of SEQ ID NO: 153, and the light chain CDR3 of SEQ IDNO: 169. In another specific embodiment, an antibody of the inventioncomprises a heavy chain variable region comprising a heavy chain CDR1selected from the group of SEQ ID NO: 3, SEQ ID NO: 13, and SEQ ID NO:23; a heavy chain CDR2 selected from the group of SEQ ID NO: 35, SEQ IDNO: 69, and SEQ ID NO: 101; and the heavy chain CDR3 of SEQ ID NO: 137,and a light chain variable region comprising the light chain CDR1 of SEQID NO: 149, the light chain CDR2 of SEQ ID NO: 157, and the light chainCDR3 of SEQ ID NO: 167. In another specific embodiment, an antibody ofthe invention comprises a heavy chain variable region comprising a heavychain CDR1 selected from the group of SEQ ID NO: 3, SEQ ID NO: 7, SEQ IDNO: 13, SEQ ID NO: 17, SEQ ID NO: 23, and SEQ ID NO: 29; a heavy chainCDR2 selected from the group of SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO:47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ IDNO: 57, SEQ ID NO: 59, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 77, SEQID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87,SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 97, SEQ ID NO:109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO:129, and SEQ ID NO: 131; and the heavy chain CDR3 of SEQ ID NO: 135, anda light chain variable region comprising the light chain CDR1 of SEQ IDNO: 143, the light chain CDR2 of SEQ ID NO: 151, and the light chainCDR3 of SEQ ID NO: 163. In a further specific embodiment, an antibody ofthe invention comprises a heavy chain variable region comprising a heavychain CDR1 selected from the group of SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 19, SEQ ID NO:21, SEQ ID NO: 31, and SEQ ID NO: 33; a heavy chainCDR2 selected from the group of SEQ ID NO: 61, SEQ ID NO: 67, SEQ ID NO:95, SEQ ID NO: 99, SEQ ID NO: 127, and SEQ ID NO: 133; and the heavychain CDR3 of SEQ ID NO: 137, and a light chain variable regioncomprising the light chain CDR1 of SEQ ID NO: 147, the light chain CDR2of SEQ ID NO: 155, and the light chain CDR3 of SEQ ID NO: 167. In afurther specific embodiment, an antibody of the invention comprises aheavy chain variable region comprising a heavy chain CDR1 selected fromthe group of SEQ ID NO: 3, SEQ ID NO: 13, and SEQ ID NO: 23; a heavychain CDR2 selected from the group of SEQ ID NO: 35, SEQ ID NO: 69, andSEQ ID NO: 101; and the heavy chain CDR3 of SEQ ID NO: 135, and a lightchain variable region comprising the light chain CDR1 of SEQ ID NO: 143,the light chain CDR2 of SEQ ID NO: 151, and the light chain CDR3 of SEQID NO: 177. In a further specific embodiment, an antibody of theinvention comprises a heavy chain variable region comprising a heavychain CDR1 selected from the group of SEQ ID NO: 3, SEQ ID NO: 13, andSEQ ID NO: 23; a heavy chain CDR2 selected from the group of SEQ ID NO:43, SEQ ID NO: 77, and SEQ ID NO: 109; and the heavy chain CDR3 of SEQID NO: 135, and a light chain variable region comprising the light chainCDR1 of SEQ ID NO: 143, the light chain CDR2 of SEQ ID NO: 151, and thelight chain CDR3 of SEQ ID NO: 163. In a further specific embodiment, anantibody of the invention comprises a heavy chain variable regioncomprising a heavy chain CDR1 selected from the group of SEQ ID NO: 3,SEQ ID NO: 13, and SEQ ID NO: 23; a heavy chain CDR2 selected from thegroup of SEQ ID NO: 45, SEQ ID NO: 79, and SEQ ID NO: 111; and the heavychain CDR3 of SEQ ID NO: 135, and a light chain variable regioncomprising the light chain CDR1 of SEQ ID NO: 143, the light chain CDR2of SEQ ID NO: 151, and the light chain CDR3 of SEQ ID NO: 163. In afurther specific embodiment, an antibody of the invention comprises aheavy chain variable region comprising a heavy chain CDR1 selected fromthe group of SEQ ID NO: 3, SEQ ID NO: 13, and SEQ ID NO: 23; a heavychain CDR2 selected from the group of SEQ ID NO: 65, SEQ ID NO: 89, andSEQ ID NO: 131; and the heavy chain CDR3 of SEQ ID NO: 135, and a lightchain variable region comprising the light chain CDR1 of SEQ ID NO: 143,the light chain CDR2 of SEQ ID NO: 151, and the light chain CDR3 of SEQID NO: 163. In a further specific embodiment, an antibody of theinvention comprises a heavy chain variable region comprising a heavychain CDR1 selected from the group of SEQ ID NO: 3, SEQ ID NO: 13, andSEQ ID NO: 23; a heavy chain CDR2 selected from the group of SEQ ID NO:47, SEQ ID NO: 81, and SEQ ID NO: 113; and the heavy chain CDR3 of SEQID NO: 135, and a light chain variable region comprising the light chainCDR1 of SEQ ID NO: 143, the light chain CDR2 of SEQ ID NO: 151, and thelight chain CDR3 of SEQ ID NO: 163. In a further specific embodiment, anantibody of the invention comprises a heavy chain variable regioncomprising a heavy chain CDR1 selected from the group of SEQ ID NO: 9,SEQ ID NO: 19, and SEQ ID NO: 31; a heavy chain CDR2 selected from thegroup of SEQ ID NO: 61, SEQ ID NO: 95, and SEQ ID NO: 127; and the heavychain CDR3 of SEQ ID NO: 137, and a light chain variable regioncomprising the light chain CDR1 of SEQ ID NO: 147, the light chain CDR2of SEQ ID NO: 155, and the light chain CDR3 of SEQ ID NO: 167.

In one embodiment, an antibody of the invention comprises a heavy chainvariable region (VH) comprising an amino acid sequence having at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to asequence selected from the group of SEQ ID NO: 197, SEQ ID NO: 201, SEQID NO: 203, SEQ ID NO: 207, SEQ ID NO: 211, SEQ ID NO: 215, SEQ ID NO:219, SEQ ID NO: 223, SEQ ID NO: 227, SEQ ID NO: 231, SEQ ID NO: 235, SEQID NO: 239, SEQ ID NO: 243, SEQ ID NO: 247, SEQ ID NO: 251, SEQ ID NO:255, SEQ ID NO: 259, SEQ ID NO: 263, SEQ ID NO: 267, SEQ ID NO: 271, SEQID NO: 275, SEQ ID NO: 279, SEQ ID NO: 283, SEQ ID NO: 287, SEQ ID NO:291, SEQ ID NO: 295, SEQ ID NO: 299, SEQ ID NO: 303, SEQ ID NO: 307, andSEQ ID NO: 311. In one embodiment, the antibody comprises a heavy chainvariable region comprising an amino acid sequence selected from thegroup of SEQ ID NO: 197, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 207,SEQ ID NO: 211, SEQ ID NO: 215, SEQ ID NO: 219, SEQ ID NO: 223, SEQ IDNO: 227, SEQ ID NO: 231, SEQ ID NO: 235, SEQ ID NO: 239, SEQ ID NO: 243,SEQ ID NO: 247, SEQ ID NO: 251, SEQ ID NO: 255, SEQ ID NO: 259, SEQ IDNO: 263, SEQ ID NO: 267, SEQ ID NO: 271, SEQ ID NO: 275, SEQ ID NO: 279,SEQ ID NO: 283, SEQ ID NO: 287, SEQ ID NO: 291, SEQ ID NO: 295, SEQ IDNO: 299, SEQ ID NO: 303, SEQ ID NO: 307, and SEQ ID NO: 311.

In certain embodiments, a VH sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions(e.g., conservative substitutions), insertions, or deletions relative tothe reference sequence, but an anti-FAP antibody comprising thatsequence retains the ability to bind to FAP. In certain embodiments, atotal of 1 to 10 amino acids have been substituted, inserted and/ordeleted in SEQ ID NO 197, 201, 203, 207, 211, 215, 219, 223, 227, 231,235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287,291, 295, 299, 303, 307 or 311. In certain embodiments, substitutions,insertions, or deletions occur in regions outside the HVRs or CDRs(i.e., in the FRs). Optionally, an anti-FAP antibody according to theinvention comprises the VH sequence in SEQ ID NO 197, 201, 203, 207,211, 215, 219, 223, 227, 231, 235, 239, 243, 247, 251, 255, 259, 263,267, 271, 275, 279, 283, 287, 291, 295, 299, 303, 307 or 311, includingpost-translational modifications of that sequence. In a particularembodiment, the VH comprises one, two or three heavy chain CDRs selectedfrom the sequences set forth in SEQ ID NOs 3, 5, 7, 9, 11, 13, 15, 17,19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53,55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89,91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119,121, 123, 125, 127, 129, 131, 133, 135, 137, 139 and 141 for the HCDR1,HCDR2 and HCDR3.

In another embodiment, an antibody of the invention comprises a lightchain variable region comprising an amino acid sequence having at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to asequence selected from the group of SEQ ID NO: 193, SEQ ID NO: 195, SEQID NO: 199, SEQ ID NO: 205, SEQ ID NO: 209, SEQ ID NO: 213, SEQ ID NO:217, SEQ ID NO: 221, SEQ ID NO: 225, SEQ ID NO: 229, SEQ ID NO: 233, SEQID NO: 237, SEQ ID NO: 241, SEQ ID NO: 245, SEQ ID NO: 249, SEQ ID NO:253, SEQ ID NO: 257, SEQ ID NO: 261, SEQ ID NO: 265, SEQ ID NO: 269, SEQID NO: 273, SEQ ID NO: 277, SEQ ID NO: 281, SEQ ID NO: 285, SEQ ID NO:289, SEQ ID NO: 293, SEQ ID NO: 297, SEQ ID NO: 301, SEQ ID NO: 305, andSEQ ID NO: 309. In yet another embodiment, the antibody comprises alight chain variable region comprising an amino acid sequence selectedfrom the group of: SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 199, SEQID NO: 205, SEQ ID NO: 209, SEQ ID NO: 213, SEQ ID NO: 217, SEQ ID NO:221, SEQ ID NO: 225, SEQ ID NO: 229, SEQ ID NO: 233, SEQ ID NO: 237, SEQID NO: 241, SEQ ID NO: 245, SEQ ID NO: 249, SEQ ID NO: 253, SEQ ID NO:257, SEQ ID NO: 261, SEQ ID NO: 265, SEQ ID NO: 269, SEQ ID NO: 273, SEQID NO: 277, SEQ ID NO: 281, SEQ ID NO: 285, SEQ ID NO: 289, SEQ ID NO:293, SEQ ID NO: 297, SEQ ID NO: 301, SEQ ID NO: 305, and SEQ ID NO: 309.

In certain embodiments, a VL sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identity contains substitutions(e.g., conservative substitutions), insertions, or deletions relative tothe reference sequence, but an anti-FAP antibody comprising thatsequence retains the ability to bind to FAP. In certain embodiments, atotal of 1 to 10 amino acids have been substituted, inserted and/ordeleted in SEQ ID NO 193, 195, 199, 205, 209, 213, 217, 221, 225, 229,233, 237, 241, 245, 249, 253, 257, 261, 265, 269, 273, 277, 281, 285,289, 293, 297, 301, 305 or 309. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs or CDRs (i.e., in the FRs). Optionally, an anti-FAP antibody of theinvention comprises the VL sequence in SEQ ID NO 193, 195, 199, 205,209, 213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261,265, 269, 273, 277, 281, 285, 289, 293, 297, 301, 305 or 309, includingpost-translational modifications of that sequence. In a particularembodiment, the VL comprises one, two or three light chain CDRs selectedfrom sequences set forth in SEQ ID NOs 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175 and 177 for theLCDR1, LCDR2 and LCDR3.

In another aspect, an anti-FAP antibody is provided, wherein theantibody comprises a VH as in any of the embodiments provided above, anda VL as in any of the embodiments provided above. In one embodiment, theantibody comprises a heavy chain variable region comprising an aminoacid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identical to a sequence selected from the group ofSEQ ID NO: 197, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 207, SEQ IDNO: 211, SEQ ID NO: 215, SEQ ID NO: 219, SEQ ID NO: 223, SEQ ID NO: 227,SEQ ID NO: 231, SEQ ID NO: 235, SEQ ID NO: 239, SEQ ID NO: 243, SEQ IDNO: 247, SEQ ID NO: 251, SEQ ID NO: 255, SEQ ID NO: 259, SEQ ID NO: 263,SEQ ID NO: 267, SEQ ID NO: 271, SEQ ID NO: 275, SEQ ID NO: 279, SEQ IDNO: 283, SEQ ID NO: 287, SEQ ID NO: 291, SEQ ID NO: 295, SEQ ID NO: 299,SEQ ID NO: 303, SEQ ID NO: 307, and SEQ ID NO: 311, and a light chainvariable region comprising an amino acid sequence that is at least about90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to asequence selected from the group of: SEQ ID NO: 193, SEQ ID NO: 195, SEQID NO: 199, SEQ ID NO: 205, SEQ ID NO: 209, SEQ ID NO: 213, SEQ ID NO:217, SEQ ID NO: 221, SEQ ID NO: 225, SEQ ID NO: 229, SEQ ID NO: 233, SEQID NO: 237, SEQ ID NO: 241, SEQ ID NO: 245, SEQ ID NO: 249, SEQ ID NO:253, SEQ ID NO: 257, SEQ ID NO: 261, SEQ ID NO: 265, SEQ ID NO: 269, SEQID NO: 273, SEQ ID NO: 277, SEQ ID NO: 281, SEQ ID NO: 285, SEQ ID NO:289, SEQ ID NO: 293, SEQ ID NO: 297, SEQ ID NO: 301, SEQ ID NO: 305, andSEQ ID NO: 309. In one embodiment, the antibody comprises the VH and VLsequences in SEQ ID NO 197, 201, 203, 207, 211, 215, 219, 223, 227, 231,235, 239, 243, 247, 251, 255, 259, 263, 267, 271, 275, 279, 283, 287,291, 295, 299, 303, 307 or 311, and SEQ ID NO 193, 195, 199, 205, 209,213, 217, 221, 225, 229, 233, 237, 241, 245, 249, 253, 257, 261, 265,269, 273, 277, 281, 285, 289, 293, 297, 301, 305 or 309, respectively,including post-translational modifications of those sequences.

In one embodiment, the antibody comprises a heavy chain variable regioncomprising an amino acid sequence selected from the group of SEQ ID NO:197, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 207, SEQ ID NO: 211, SEQID NO: 215, SEQ ID NO: 219, SEQ ID NO: 223, SEQ ID NO: 227, SEQ ID NO:231, SEQ ID NO: 235, SEQ ID NO: 239, SEQ ID NO: 243, SEQ ID NO: 247, SEQID NO: 251, SEQ ID NO: 255, SEQ ID NO: 259, SEQ ID NO: 263, SEQ ID NO:267, SEQ ID NO: 271, SEQ ID NO: 275, SEQ ID NO: 279, SEQ ID NO: 283, SEQID NO: 287, SEQ ID NO: 291, SEQ ID NO: 295, SEQ ID NO: 299, SEQ ID NO:303, SEQ ID NO: 307, and SEQ ID NO: 311, and a light chain variableregion comprising an amino acid sequence selected from the group of: SEQID NO: 193, SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 205, SEQ ID NO:209, SEQ ID NO: 213, SEQ ID NO: 217, SEQ ID NO: 221, SEQ ID NO: 225, SEQID NO: 229, SEQ ID NO: 233, SEQ ID NO: 237, SEQ ID NO: 241, SEQ ID NO:245, SEQ ID NO: 249, SEQ ID NO: 253, SEQ ID NO: 257, SEQ ID NO: 261, SEQID NO: 265, SEQ ID NO: 269, SEQ ID NO: 273, SEQ ID NO: 277, SEQ ID NO:281, SEQ ID NO: 285, SEQ ID NO: 289, SEQ ID NO: 293, SEQ ID NO: 297, SEQID NO: 301, SEQ ID NO: 305, and SEQ ID NO: 309, wherein at least one ofsaid variable regions does not comprise an amino acid sequence selectedfrom the group of SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ IDNO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207,SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ IDNO: 217, SEQ ID NO: 219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225,SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ IDNO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243,SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ IDNO: 253, and SEQ ID NO: 255.

In one embodiment, the antibody comprises a heavy chain variable regioncomprising an amino acid sequence selected from the group of SEQ ID NO:197, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 207, SEQ ID NO: 211, SEQID NO: 215, SEQ ID NO: 219, SEQ ID NO: 223, SEQ ID NO: 227, SEQ ID NO:231, SEQ ID NO: 235, SEQ ID NO: 239, SEQ ID NO: 243, SEQ ID NO: 247, SEQID NO: 251, SEQ ID NO: 255, SEQ ID NO: 259, SEQ ID NO: 263, SEQ ID NO:267, SEQ ID NO: 271, SEQ ID NO: 275, SEQ ID NO: 279, SEQ ID NO: 283, SEQID NO: 287, SEQ ID NO: 291, SEQ ID NO: 295, SEQ ID NO: 299, SEQ ID NO:303, SEQ ID NO: 307, and SEQ ID NO: 311, and a light chain variableregion comprising an amino acid selected from the group of: SEQ ID NO:193, SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 205, SEQ ID NO: 209, SEQID NO: 213, SEQ ID NO: 217, SEQ ID NO: 221, SEQ ID NO: 225, SEQ ID NO:229, SEQ ID NO: 233, SEQ ID NO: 237, SEQ ID NO: 241, SEQ ID NO: 245, SEQID NO: 249, SEQ ID NO: 253, SEQ ID NO: 257, SEQ ID NO: 261, SEQ ID NO:265, SEQ ID NO: 269, SEQ ID NO: 273, SEQ ID NO: 277, SEQ ID NO: 281, SEQID NO: 285, SEQ ID NO: 289, SEQ ID NO: 293, SEQ ID NO: 297, SEQ ID NO:301, SEQ ID NO: 305, and SEQ ID NO: 309, wherein at least one of saidvariable regions comprises an amino acid sequence selected from thegroup of SEQ ID NO: 259, SEQ ID NO: 263, SEQ ID NO: 267, SEQ ID NO: 271,SEQ ID NO: 275, SEQ ID NO:279, SEQ ID NO:283, SEQ ID NO: 287, SEQ ID NO:291, SEQ ID NO: 293, SEQ ID NO: 299, SEQ ID NO: 303, SEQ ID NO: 307, andSEQ ID NO: 311.

In one embodiment, the antibody comprises a heavy chain variable regioncomprising an amino acid sequence that is at least about 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequenceselected from the group of SEQ ID NO: 197, SEQ ID NO: 201, SEQ ID NO:203, SEQ ID NO: 207, SEQ ID NO: 211, SEQ ID NO: 215, SEQ ID NO: 219, SEQID NO: 223, SEQ ID NO: 227, SEQ ID NO: 231, SEQ ID NO: 235, SEQ ID NO:239, SEQ ID NO: 243, SEQ ID NO: 247, SEQ ID NO: 251, and SEQ ID NO: 255,and a light chain variable region comprising an amino acid sequence thatis at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or100% identical to a sequence selected from the group of: SEQ ID NO: 193,SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 205, SEQ ID NO: 209, SEQ IDNO: 213, SEQ ID NO: 217, SEQ ID NO: 221, SEQ ID NO: 225, SEQ ID NO: 229,SEQ ID NO: 233, SEQ ID NO: 237, SEQ ID NO: 241, SEQ ID NO: 245, SEQ IDNO: 249, and SEQ ID NO: 253.

In a specific embodiment, an antibody of the invention comprises a heavychain variable region comprising the amino acid sequence of SEQ IDNO:197, and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 193 or SEQ ID NO: 195. In another specificembodiment, an antibodies of the invention comprises a heavy chainvariable region comprising the amino acid sequence of SEQ ID NO: 201 orSEQ ID NO: 203, and a light chain variable region comprising the aminoacid sequence of SEQ ID NO: 199. In yet another specific embodiment anantibody of the invention comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 207, and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 205. Inanother specific embodiment, an antibodies of the invention comprises aheavy chain variable region comprising the amino acid sequence of SEQ IDNO: 211, and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 209. In yet another specific embodiment anantibody of the invention comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 219, and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 217. Inanother embodiment, an antibody of the invention comprises a heavy chainvariable region comprising an amino acid sequence selected from thegroup of SEQ ID NO: 259, SEQ ID NO: 263, SEQ ID NO: 267, SEQ ID NO: 271,SEQ ID NO: 275, SEQ ID NO:279, SEQ ID NO:283, SEQ ID NO: 287, SEQ ID NO:291, SEQ ID NO: 299, SEQ ID NO: 303, SEQ ID NO: 307, and SEQ ID NO: 311,or a light chain variable region comprising the amino acid sequence ofSEQ ID NO: 293. In a specific embodiment, the antibodies of theinvention comprise a) a heavy chain variable region comprising an aminoacid sequence selected from SEQ ID NO: 259, SEQ ID NO: 263, SEQ ID NO:267, SEQ ID NO: 271, SEQ ID NO: 275, SEQ ID NO:279, SEQ ID NO:283, SEQID NO: 287, SEQ ID NO: 291, SEQ ID NO: 303, and SEQ ID NO: 307, and alight chain variable region comprising the amino acid sequence of SEQ IDNO: 195, or b) a heavy chain variable region comprising the amino acidsequence or SEQ ID NO: 299 or SEQ ID NO: 311, and a light chain variableregion comprising the amino acid sequence of SEQ ID NO: 205, or c) aheavy chain variable region comprising the amino acid sequence or SEQ IDNO: 197, and a light chain variable region comprising the amino acidsequence of SEQ ID NO: 293. In a specific embodiment, an antibody of theinvention comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 259 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 195. In anotherspecific embodiment, an antibodies of the invention comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:263 and a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 195. In a specific embodiment, an antibody of theinvention comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 307 and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 305. In anotherspecific embodiment, an antibodies of the invention comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:267 and a light chain variable region comprising the amino acid sequenceof SEQ ID NO: 265. In yet another specific embodiment an antibody of theinvention comprises a heavy chain variable region comprising the aminoacid sequence of SEQ ID NO: 299, and a light chain variable regioncomprising the amino acid sequence of SEQ ID NO: 205. In a particularembodiment, the antibody according to any of the above embodimentsadditionally comprises an Fc region or a region equivalent to the Fcregion of an immunoglobulin.

In one embodiment an antibody of the invention comprises an Fc region,particularly a IgG Fc region, most particularly a IgG1 Fc region.

In a particular embodiment, the antibody of the invention is a fulllength antibody, particularly an IgG class antibody, most particularlyan IgG1 isotype antibody. In another embodiment, the antibody of theinvention is an antibody fragment, selected from the group of: an scFvfragment, an Fv fragment, a Fab fragment, and a F(ab′)2 fragment. In afurther embodiment, the antibody of the invention is an antibodyfragment having an Fc region, or a fusion protein that comprises aregion equivalent to the Fc region of an immunoglobulin. In oneembodiment, the antibody of the invention is a monoclonal antibody.

In one embodiment, an antibody of the invention is chimeric, morespecifically humanized. In a particular embodiment, an antibody of theinvention is human. In another embodiment, an antibody of the inventioncomprises a human constant region. In one embodiment the antibody of theinvention comprises a human Fc region, particularly a human IgG Fcregion, most particularly a human IgG1 Fc region.

In one embodiment, an antibody of the invention comprises a heavy chainconstant region, wherein said heavy chain constant region is a human IgGconstant region, particularly a human IgG1 constant region, comprisingan Fc region. In a specific embodiment, the antibody comprises a heavychain constant region comprising the amino acid sequence of SEQ ID NO:313. In another specific embodiment an antibody of the inventioncomprises a light chain constant region comprising the amino acidsequence of SEQ ID NO: 315. In yet another specific embodiment, anantibody of the invention comprises a heavy chain constant regioncomprising the amino acid sequence of SEQ ID NO: 313, and a light chainconstant region comprising the amino acid sequence of SEQ ID NO: 315.

In a particular embodiment, the invention provides an antibody thatspecifically binds to FAP, wherein said antibody comprises a) a heavychain variable region comprising an amino acid sequence that is at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identicalto a sequence selected from the group of SEQ ID NO: 197, SEQ ID NO: 201,SEQ ID NO: 203, SEQ ID NO: 207, SEQ ID NO: 211, SEQ ID NO: 215, SEQ IDNO: 219, SEQ ID NO: 223, SEQ ID NO: 227, SEQ ID NO: 231, SEQ ID NO: 235,SEQ ID NO: 239, SEQ ID NO: 243, SEQ ID NO: 247, SEQ ID NO: 251, SEQ IDNO: 255, SEQ ID NO: 259, SEQ ID NO: 263, SEQ ID NO: 267, SEQ ID NO: 271,SEQ ID NO: 275, SEQ ID NO: 279, SEQ ID NO: 283, SEQ ID NO: 287, SEQ IDNO: 291, SEQ ID NO: 295, SEQ ID NO: 299, SEQ ID NO: 303, SEQ ID NO: 307,and SEQ ID NO: 311, or a light chain variable region comprising an aminoacid sequence that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or 100% identical to a sequence selected from the group ofSEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 205, SEQ IDNO: 209, SEQ ID NO: 213, SEQ ID NO: 217, SEQ ID NO: 221, SEQ ID NO: 225,SEQ ID NO: 229, SEQ ID NO: 233, SEQ ID NO: 237, SEQ ID NO: 241, SEQ IDNO: 245, SEQ ID NO: 249, SEQ ID NO: 253, SEQ ID NO: 257, SEQ ID NO: 261,SEQ ID NO: 265, SEQ ID NO: 269, SEQ ID NO: 273, SEQ ID NO: 277, SEQ IDNO: 281, SEQ ID NO: 285, SEQ ID NO: 289, SEQ ID NO: 293, SEQ ID NO: 297,SEQ ID NO: 301, SEQ ID NO: 305, and SEQ ID NO: 309, or a combinationthereof, and b) an Fc region or a region equivalent to the Fc region ofan immunoglobulin.

In one embodiment, an antibody of the invention comprises an Fc region,wherein said Fc region is a glycoengineered Fc region. In a furtherembodiment, an antibody of the invention is glycoengineered to havemodified oligosaccharides in the Fc region. In a specific embodiment,the antibody has an increased proportion of bisected oligosaccharides inthe Fc region, compared to a non-glycoengineered antibody. In a morespecific embodiment, at least about 10%, about 15%, about 20%, about25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%,about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about90%, about 95%, or about 100%, preferably at least about 50%, morepreferably at least about 70%, of the N-linked oligosaccharides in theFc region of the antibody are bisected. The bisected oligosaccharidesmay be of the hybrid or complex type.

In another specific embodiment, an antibody of the invention has anincreased proportion of non-fucosylated oligosaccharides in the Fcregion, compared to a non-glycoengineered antibody. In a more specificembodiment, at least about 20%, about 25%, about 30%, about 35%, about40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%,preferably at least about 50%, more preferably at least about 70%, ofthe N-linked oligosaccharides in the Fc region of the antibody arenon-fucosylated. The non-fucosylated oligosaccharides may be of thehybrid or complex type.

In a particular embodiment, an antibody of the invention has anincreased proportion of bisected, non-fucosylated oligosaccharides inthe Fc region, compared to a non-glycoengineered antibody. Specifically,the antibody comprises an Fc region in which at least about 10%, about15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or about 100%, preferably at leastabout 15%, more preferably at least about 25%, at least about 35% or atleast about 50%, of the N-linked oligosaccharides are bisected,non-fucosylated. The bisected, non-fucosylated oligosaccharides may beof the hybrid or complex type.

In one embodiment, an antibody of the invention has increased effectorfunction and/or increased Fc receptor binding affinity. Increasedeffector function and/or increased Fc receptor binding can result e.g.from glycoengineering and/or affinity maturation of antibodies. In oneembodiment, the increased effector function and/or increased Fc receptorbinding is a result of glycoengineering of the Fc region of theantibody. In another embodiment, the increased effector function and/orincreased Fc receptor binding is a result of a combination of increasedaffinity and glycoengineering. The increased effector function caninclude, but is not limited to, one or more of the following: increasedFc-mediated cellular cytotoxicity (including increasedantibody-dependent cell-mediated cytotoxicity (ADCC)), increasedantibody-dependent cellular phagocytosis (ADCP), increased cytokinesecretion, increased immune-complex-mediated antigen uptake byantigen-presenting cells, increased binding to NK cells, increasedbinding to macrophages, increased binding to monocytes, increasedbinding to polymorphonuclear cells, increased direct signaling inducingapoptosis, increased crosslinking of target-bound antibodies, increaseddendritic cell maturation, or increased T cell priming. In a particularembodiment, the increased effector function is increased ADCC. Theincreased Fc receptor binding preferably is increased binding to anactivating Fc receptor, most preferably FcγRIIIa.

In one embodiment, an antibody of the invention does not cause aclinically significant level of toxicity when administered to anindividual in a therapeutically effective amount.

In one embodiment, an antibody of the invention is affinity matured. Ina further embodiment, an antibody of the invention binds to theFibroblast Activation Protein with a dissociation constant (K_(D)) valuelower than about 1 μM to about 0.001 nM, particularly a K_(D) valuelower than about 100 nM, lower than about 10 nM, lower than about 1 nM,or lower than about 0.1 nM. In one embodiment, an antibody of theinvention binds to human, mouse and cynomolgus FAP. In one embodiment,an antibody of the invention binds to human and cynomolgus FAP. In amore specific embodiment, an antibody of the invention binds to human,mouse and cynomolgus FAP with a K_(D) value lower than about 200 nM,lower than about 100 nM, more particularly lower than about 10 nM orlower than about 1 nM, most particularly lower than 0.1 nM. K_(D) valuesare determined by Surface Plasmon Resonance, using the antibodies as Fabor IgG, particularly as IgG.

In one embodiment, an anti-FAP antibody of the invention binds FAP inhuman tissues. In one embodiment an anti-FAP antibody of the inventionis cross-reactive for human and murine FAP. In another embodiment, anantibody of the invention has no substantial cross-reactivity to othermembers of the dipeptidyl peptidase IV family, in particular toDPPIV/CD26. In one embodiment, an anti-FAP antibody of the inventiondoes not induce internalization of FAP upon binding of said antibody toFAP expressed on the surface of a cell.

In a particular embodiment, the invention provides an antibody thatspecifically binds to FAP, wherein said antibody comprises a heavy chainvariable region comprising an amino acid sequence selected from thegroup of SEQ ID NO: 197, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 207,SEQ ID NO: 211, SEQ ID NO: 215, SEQ ID NO: 219, SEQ ID NO: 223, SEQ IDNO: 227, SEQ ID NO: 231, SEQ ID NO: 235, SEQ ID NO: 239, SEQ ID NO: 243,SEQ ID NO: 247, SEQ ID NO: 251, SEQ ID NO: 255, SEQ ID NO: 259, SEQ IDNO: 263, SEQ ID NO: 267, SEQ ID NO: 271, SEQ ID NO: 275, SEQ ID NO: 279,SEQ ID NO: 283, SEQ ID NO: 287, SEQ ID NO: 291, SEQ ID NO: 295, SEQ IDNO: 299, SEQ ID NO: 303, SEQ ID NO: 307, and SEQ ID NO: 311, a lightchain variable region comprising an amino acid sequence selected fromthe group of SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO:205, SEQ ID NO: 209, SEQ ID NO: 213, SEQ ID NO: 217, SEQ ID NO: 221, SEQID NO: 225, SEQ ID NO: 229, SEQ ID NO: 233, SEQ ID NO: 237, SEQ ID NO:241, SEQ ID NO: 245, SEQ ID NO: 249, SEQ ID NO: 253, SEQ ID NO: 257, SEQID NO: 261, SEQ ID NO: 265, SEQ ID NO: 269, SEQ ID NO: 273, SEQ ID NO:277, SEQ ID NO: 281, SEQ ID NO: 285, SEQ ID NO: 289, SEQ ID NO: 293, SEQID NO: 297, SEQ ID NO: 301, SEQ ID NO: 305, and SEQ ID NO: 309, and ahuman IgG Fc region, and wherein optionally said antibody isglycoengineered to have increased effector function and/or Fc receptorbinding affinity. In another particular embodiment, the inventionprovides an antibody that specifically binds to FAP, wherein saidantibody comprises a heavy chain variable region comprising an aminoacid sequence selected from the group of SEQ ID NO: 197, SEQ ID NO: 201,SEQ ID NO: 203, SEQ ID NO: 207, SEQ ID NO: 211, SEQ ID NO: 215, SEQ IDNO: 219, SEQ ID NO: 223, SEQ ID NO: 227, SEQ ID NO: 231, SEQ ID NO: 235,SEQ ID NO: 239, SEQ ID NO: 243, SEQ ID NO: 247, SEQ ID NO: 251, SEQ IDNO: 255, SEQ ID NO: 259, SEQ ID NO: 263, SEQ ID NO: 267, SEQ ID NO: 271,SEQ ID NO: 275, SEQ ID NO: 279, SEQ ID NO: 283, SEQ ID NO: 287, SEQ IDNO: 291, SEQ ID NO: 295, SEQ ID NO: 299, SEQ ID NO: 303, SEQ ID NO: 307,and SEQ ID NO: 311, a light chain variable region comprising an aminoacid sequence selected from the group of SEQ ID NO: 193, SEQ ID NO: 195,SEQ ID NO: 199, SEQ ID NO: 205, SEQ ID NO: 209, SEQ ID NO: 213, SEQ IDNO: 217, SEQ ID NO: 221, SEQ ID NO: 225, SEQ ID NO: 229, SEQ ID NO: 233,SEQ ID NO: 237, SEQ ID NO: 241, SEQ ID NO: 245, SEQ ID NO: 249, SEQ IDNO: 253, SEQ ID NO: 257, SEQ ID NO: 261, SEQ ID NO: 265, SEQ ID NO: 269,SEQ ID NO: 273, SEQ ID NO: 277, SEQ ID NO: 281, SEQ ID NO: 285, SEQ IDNO: 289, SEQ ID NO: 293, SEQ ID NO: 297, SEQ ID NO: 301, SEQ ID NO: 305,and SEQ ID NO: 309, and a human IgG Fc region, and wherein said antibodyhas an increased proportion of non-fucosylated oligosaccharides and/oran increased proportion of bisected oligosaccharides in said Fc region.

In one aspect, the invention provides for an antibody that specificallybind to FAP, wherein said antibody is derived from a parent antibodycomprising the heavy chain CDR1 of SEQ ID NO: 3, the heavy chain CDR2 ofSEQ ID NO: 35, a heavy chain CDR3 selected from the group of SEQ ID NO:135, SEQ ID NO: 137, SEQ ID NO: 139 and SEQ ID NO: 141, the light chainCDR1 of SEQ ID NO: 145, the light chain CDR2 of SEQ ID NO: 153 and alight chain CDR3 selected from the group of SEQ ID NO: 165, SEQ ID NO:167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173 and SEQ ID NO: 175,and wherein said antibody comprises at least one amino acid substitutionor deletion in at least one heavy or light chain CDR of to the parentantibody. For example, the antibody may comprise at least one, e.g. fromabout one to about ten (i.e., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10),and particularly from about two to about five, substitutions in one ormore hypervariable regions or CDRs (i.e., 1, 2, 3, 4, 5, or 6hypervariable regions or CDRs) of the parent antibody. In certainembodiments, any one or more amino acids of the parent antibody asprovided above are substituted or deleted at the following CDRpositions:

-   -   Heavy chain CDR1 (SEQ ID NO: 3): positions 2 and 3    -   Heavy chain CDR2 (SEQ ID NO: 35): positions 1, 3, 4, 5, 6, 7, 8        and 9    -   Light chain CDR1 (SEQ ID NO: 145): positions 7, 8 and 9    -   Light chain CDR2 (SEQ ID NO: 153): positions 1, 2, 3, 4 and 5    -   Light chain CDR3 (SEQ ID NO 165, 167, 169, 171, 173, or 175):        positions 4, 5, 6, and 7

In certain embodiments, the substitutions are conservativesubstitutions, as provided herein. In certain embodiments, any one ormore of the following substitutions or deletions may be made in anycombination:

-   -   Heavy chain CDR1 (SEQ ID NO: 3): Y2F, H or S, A3T    -   Heavy chain CDR2 (SEQ ID NO: 35): A1G, S3G, I, W or L, G4V, S, A        or T, S5G or N, G6T or A, G7R, S, A, E or N, S8Y, L, R, I, N, Q,        I or deleted, T9 deleted    -   Light chain CDR1 (SEQ ID NO: 145): S7T, S8R or S9N    -   Light chain CDR2 (SEQ ID NO: 153): Y1N, I or Q, G2V, A3G, S4T or        Y, S5R, T or I    -   Light chain CDR3 (SEQ ID NO 165, 167, 169, 171, 173, or 175):        G4A, Q, N, L or H5I, L, V, Q, N or I6M, I7L

Additionally, the antibodies may also comprise one or more additions,deletions and/or substitutions in one or more framework regions ofeither the heavy or the light chain, compared to the parent antibody. Inone embodiment, said at least one amino acid substitution in at leastone CDR contributes to increased binding affinity of the antibodycompared to its parent antibody. In another embodiment said antibody hasat least about 2-fold to about 10-fold greater affinity for FAP than theparent antibody (when comparing the antibody of the invention and theparent antibody in the same format, e.g. the Fab format). Further, theantibody derived from a parent antibody may incorporate any of thefeatures, singly or in combination, described in the precedingparagraphs in relation to the antibodies of the invention.

The present invention also provides for polynucleotides encodingantibodies that specifically bind to FAP. In one aspect, the inventionis directed to an isolated polynucleotide encoding a polypeptide thatforms part of an anti-FAP antibody according to the invention asdescribed hereinbefore. In one embodiment, the isolated polynucleotideencodes an antibody heavy chain and/or an antibody light chain thatforms part of an anti-FAP antibody according to the invention asdescribed hereinbefore.

In one embodiment, the invention is directed to an isolatedpolynucleotide comprising a sequence encoding one or more (e.g. one,two, three, four, five, or six) of the heavy or light chaincomplementarity determining regions (CDRs) set forth in SEQ ID NOs 3, 5,7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77,79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 171, 173, 175 and 177, or a variant or truncated form thereofcontaining at least the specificity-determining residues (SDRs) for saidCDR. In another embodiment, the polynucleotide comprises a sequence thatencodes three heavy chain CDRs (e.g., HCDR1, HCDR2, and HCDR3) or threelight chain CDRs (e.g. LCDR1, LCDR2, and LCDR3) selected from SEQ ID NOs3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109,111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137,139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165,167, 169, 171, 173, 175 and 177, or variants or truncated forms thereofcontaining at least the SDRs for each of said three complementaritydetermining regions. In yet another embodiment, the polynucleotidecomprises a sequence encoding three heavy chain CDRs (e.g., HCDR1,HCDR2, and HCDR3) and three light chain CDRs (e.g. LCDR1, LCDR2, andLCDR3) selected from SEQ ID NOs 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59,61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95,97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125,127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153,155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175 and 177. In aparticular embodiment the polynucleotide encoding one or more CDRscomprises a sequence that is at least about 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to one or more of the CDR nucleotide sequencesshown in SEQ ID NOs 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30,32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128,130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156,158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 179, 180, 181,182, 183, 184, 185, 186, 187, 188, 189, 190, 191 and 192.

In a further embodiment, the polynucleotide comprises a sequenceencoding a heavy chain variable region selected from the group of SEQ IDNO: 197, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 207, SEQ ID NO: 211,SEQ ID NO: 215, SEQ ID NO: 219, SEQ ID NO: 223, SEQ ID NO: 227, SEQ IDNO: 231, SEQ ID NO: 235, SEQ ID NO: 239, SEQ ID NO: 243, SEQ ID NO: 247,SEQ ID NO: 251, SEQ ID NO: 255, SEQ ID NO: 259, SEQ ID NO: 263, SEQ IDNO: 267, SEQ ID NO: 271, SEQ ID NO: 275, SEQ ID NO: 279, SEQ ID NO: 283,SEQ ID NO: 287, SEQ ID NO: 291, SEQ ID NO: 295, SEQ ID NO: 299, SEQ IDNO: 303, SEQ ID NO: 307, and SEQ ID NO: 311, and/or a sequence encodinga light chain variable region selected from the group of SEQ ID NO: 193,SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 205, SEQ ID NO: 209, SEQ IDNO: 213, SEQ ID NO: 217, SEQ ID NO: 221, SEQ ID NO: 225, SEQ ID NO: 229,SEQ ID NO: 233, SEQ ID NO: 237, SEQ ID NO: 241, SEQ ID NO: 245, SEQ IDNO: 249, SEQ ID NO: 253, SEQ ID NO: 257, SEQ ID NO: 261, SEQ ID NO: 265,SEQ ID NO: 269, SEQ ID NO: 273, SEQ ID NO: 277, SEQ ID NO: 281, SEQ IDNO: 285, SEQ ID NO: 289, SEQ ID NO: 293, SEQ ID NO: 297, SEQ ID NO: 301,SEQ ID NO: 305, and SEQ ID NO: 309. In a particular embodiment, thepolynucleotide encoding a heavy chain and/or light chain variable regioncomprises a sequence selected from the group of variable regionnucleotide sequences presented in SEQ ID NOs 194, 196, 198, 200, 202,204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230,232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258,260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286,288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310 and 312, or acombination thereof.

In a specific embodiment, the polynucleotide comprises a sequenceencoding a heavy chain variable region selected from the group of SEQ IDNO: 197, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 207, SEQ ID NO: 211,SEQ ID NO: 215, SEQ ID NO: 219, SEQ ID NO: 223, SEQ ID NO: 227, SEQ IDNO: 231, SEQ ID NO: 235, SEQ ID NO: 239, SEQ ID NO: 243, SEQ ID NO: 247,SEQ ID NO: 251, SEQ ID NO: 255, SEQ ID NO: 259, SEQ ID NO: 263, SEQ IDNO: 267, SEQ ID NO: 271, SEQ ID NO: 275, SEQ ID NO: 279, SEQ ID NO: 283,SEQ ID NO: 287, SEQ ID NO: 291, SEQ ID NO: 295, SEQ ID NO: 299, SEQ IDNO: 303, SEQ ID NO: 307, and SEQ ID NO: 311, and a sequence encoding aheavy chain constant region, particularly a human heavy chain constantregion. In a particular embodiment, said heavy chain constant region isa human IgG heavy chain constant region, specifically a human IgG1 heavychain constant region, comprising an Fc region. In a specificembodiment, said heavy chain constant region comprises the sequence ofSEQ ID NO: 313. In another specific embodiment, the polynucleotidecomprises a sequence encoding a light chain variable region selectedfrom the group of SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 199, SEQ IDNO: 205, SEQ ID NO: 209, SEQ ID NO: 213, SEQ ID NO: 217, SEQ ID NO: 221,SEQ ID NO: 225, SEQ ID NO: 229, SEQ ID NO: 233, SEQ ID NO: 237, SEQ IDNO: 241, SEQ ID NO: 245, SEQ ID NO: 249, SEQ ID NO: 253, SEQ ID NO: 257,SEQ ID NO: 261, SEQ ID NO: 265, SEQ ID NO: 269, SEQ ID NO: 273, SEQ IDNO: 277, SEQ ID NO: 281, SEQ ID NO: 285, SEQ ID NO: 289, SEQ ID NO: 293,SEQ ID NO: 297, SEQ ID NO: 301, SEQ ID NO: 305, and SEQ ID NO: 309, anda sequence encoding a light chain constant region, particularly a humanlight chain constant region. In a specific embodiment, said light chainconstant region comprises the sequence of SEQ ID NO: 315.

In one embodiment, the invention is directed to a composition thatcomprises a first isolated polynucleotide encoding a polypeptidecomprising an amino acid sequence that is at least about 90%, 95%, 96%,97%, 98%, 99%, or 100% identical to a sequence selected from the groupconsisting of SEQ ID NO: 197, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO:207, SEQ ID NO: 211, SEQ ID NO: 215, SEQ ID NO: 219, SEQ ID NO: 223, SEQID NO: 227, SEQ ID NO: 231, SEQ ID NO: 235, SEQ ID NO: 239, SEQ ID NO:243, SEQ ID NO: 247, SEQ ID NO: 251, SEQ ID NO: 255, SEQ ID NO: 259, SEQID NO: 263, SEQ ID NO: 267, SEQ ID NO: 271, SEQ ID NO: 275, SEQ ID NO:279, SEQ ID NO: 283, SEQ ID NO: 287, SEQ ID NO: 291, SEQ ID NO: 295, SEQID NO: 299, SEQ ID NO: 303, SEQ ID NO: 307, and SEQ ID NO: 311, and asecond isolated polynucleotide encoding a polypeptide comprising anamino acid sequence that is at least about 90%, 95%, 96%, 97%, 98%, 99%,or 100% identical to a sequence selected from the group consisting ofSEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 199, SEQ ID NO: 205, SEQ IDNO: 209, SEQ ID NO: 213, SEQ ID NO: 217, SEQ ID NO: 221, SEQ ID NO: 225,SEQ ID NO: 229, SEQ ID NO: 233, SEQ ID NO: 237, SEQ ID NO: 241, SEQ IDNO: 245, SEQ ID NO: 249, SEQ ID NO: 253, SEQ ID NO: 257, SEQ ID NO: 261,SEQ ID NO: 265, SEQ ID NO: 269, SEQ ID NO: 273, SEQ ID NO: 277, SEQ IDNO: 281, SEQ ID NO: 285, SEQ ID NO: 289, SEQ ID NO: 293, SEQ ID NO: 297,SEQ ID NO: 301, SEQ ID NO: 305, and SEQ ID NO: 309.

In one embodiment, the invention is directed to a composition thatcomprises a first isolated polynucleotide comprising a sequence that isat least about 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to asequence selected from the group consisting of SEQ ID NO: 198, SEQ IDNO: 202, SEQ ID NO: 204, SEQ ID NO: 208, SEQ ID NO: 212, SEQ ID NO: 216,SEQ ID NO: 220, SEQ ID NO: 224, SEQ ID NO: 228, SEQ ID NO: 232, SEQ IDNO: 236, SEQ ID NO: 240, SEQ ID NO: 244, SEQ ID NO: 248, SEQ ID NO: 252,SEQ ID NO: 256, SEQ ID NO: 260, SEQ ID NO: 264, SEQ ID NO: 268, SEQ IDNO: 272, SEQ ID NO: 276, SEQ ID NO: 280, SEQ ID NO: 284, SEQ ID NO: 288,SEQ ID NO: 292, SEQ ID NO: 296, SEQ ID NO: 300, SEQ ID NO: 304, SEQ IDNO: 308, and SEQ ID NO: 312, and a second isolated polynucleotidecomprising a sequence that is at least about 90%, 95%, 96%, 97%, 98%,99%, or 100% identical to a sequence selected from the group consistingof SEQ ID NO: 194, SEQ ID NO: 196, SEQ ID NO: 200, SEQ ID NO: 206, SEQID NO: 210, SEQ ID NO: 214, SEQ ID NO: 218, SEQ ID NO: 222, SEQ ID NO:226, SEQ ID NO: 230, SEQ ID NO: 234, SEQ ID NO: 238, SEQ ID NO: 242, SEQID NO: 246, SEQ ID NO: 250, SEQ ID NO: 254, SEQ ID NO: 258, SEQ ID NO:262, SEQ ID NO: 266, SEQ ID NO: 270, SEQ ID NO: 274, SEQ ID NO: 278, SEQID NO: 282, SEQ ID NO: 286, SEQ ID NO: 290, SEQ ID NO: 294, SEQ ID NO:298, SEQ ID NO: 302, SEQ ID NO: 306, and SEQ ID NO: 310.

In a further aspect, the invention is also directed to isolatedpolypeptides, encoded by any of the polynucleotides according theinvention as described hereinbefore.

In a further aspect, an anti-FAP antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in Sections 1-6 below:

1. Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (K_(D)) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or≦0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g., from10⁻⁹M to 10⁻¹³ M). Preferably, the antibodies provided herein bind toFibroblast Activation Protein (FAP), in particular human FAP, with aK_(D) value lower than 1 nM, as determined by Surface Plasmon Resonance(SPR).

According to one embodiment, K_(D) is measured using surface plasmonresonance. Such an assay can be performed, for example, using aBIACORE®-T100 machine (GE Healthcare) at 25° C. with CM5 chips forantigen immobilization. Briefly, carboxymethylated dextran biosensorchips (CM5, GE Healthcare.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Anti-His antibody (Penta His, Qiagen) is diluted with 10 mM sodiumacetate, pH 5, to 40 μg/ml before injection at a flow rate of 10μl/minute to achieve approximately 9000 response units (RU) of coupledprotein. Following the injection of the anti-His antibody, 1 Methanolamine is injected to block unreacted groups. Subsequently,His-tagged antigen is injected at 10 μl/min at 10 nM for 20 sec (formeasurements with Fab fragments) or at 20 nM for 25 s (for measurementswith IgG antibodies) and is captured via its His tag by the immobilizedanti-His antibody. Protein and DNA sequences of suitable FAP antigenconstructs are shown in SEQ ID NOs 317-322. For kinetics measurements,serial dilutions of antibody (two-fold dilutions, range between 6.25 nMto 200 nM for Fab fragments, or five-fold dilutions, range between 3.2pM to 10 nM for IgG) are injected in 10 mM HEPES, 150 mM NaCl, 3 mMEDTA, 0.05% Surfactant P20, pH 7.4 at 25° C. at a flow rate of 90μl/min. The following parameters are applied: Association time 180 s,dissociation 300 s (for Fab) or 900 s (for IgG), regeneration with 10 mMglycine pH 2 for 60 s between each cycle. Association rates (k_(on)) anddissociation rates (k_(off)) are calculated using a simple one-to-oneLangmuir binding model (BIACORE® T100 Evaluation Software) bysimultaneously fitting the association and dissociation sensorgrams. Theequilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on.) See, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999).

2. Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003), or Carter, Nat. Rev. Immunol. 6:343-357(2006).

Single-chain Fv or scFv fragments comprise a VH domain and a VL domainas a single polypeptide chain. Typically, the VH and VL domains arejoined by a linker sequence. For a review of scFv fragments, see, e.g.,Plückthun, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90:6444-6448 (1993). Triabodies and tetrabodies arealso described in Hudson et al., Nat. Med. 9:129-134 (2003).

A minibody is a bivalent, homodimeric scFv derivative that contains aconstant region, typically the CH3 region of an immunoglobulin,preferably IgG, more preferably IgG1, as the dimerisation region.Generally, the constant region is connected to the scFv via a hingeregion and/or a linker region. Examples of minibody proteins can befound in Hu et al., Cancer Res. 56: 3055-61 (1996).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

3. Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof. In certain embodiments, a chimericantibody is a humanized antibody. Typically, a non-human antibody ishumanized to reduce immunogenicity to humans, while retaining thespecificity and affinity of the parental non-human antibody. Generally,a humanized antibody comprises one or more variable domains in whichHVRs, e.g., CDRs, (or portions thereof) are derived from a non-humanantibody, and FRs (or portions thereof) are derived from human antibodysequences. A humanized antibody optionally will also comprise at least aportion of a human constant region.

In some embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity. Humanization may beachieved by various methods including, but not limited to (a) graftingthe entire non-human variable domains onto human constant regions togenerate chimeric antibodies, (b) grafting only the non-human (e.g.,donor antibody) CDRs onto human (e.g., recipient antibody) framework andconstant regions with or without retention of critical frameworkresidues (e.g., those that are important for retaining good antigenbinding affinity or antibody functions), (c) grafting only the non-humanspecificity-determining regions (SDRs or a-CDRs; the residues criticalfor the antibody-antigen interaction) onto human framework and constantregions, or (d) transplanting the entire non-human variable domains, but“cloaking” them with a human-like section by replacement of surfaceresidues. Humanized antibodies and methods of making them are reviewed,e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), andare further described, e.g., in Riechmann et al., Nature 332:323-329(1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989);U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Jones etal., Nature 321:522-525 (1986); Morrison et al., Proc. Natl. Acad. Sci.81:6851-6855 (1984); Morrison and Oi, Adv. Immunol. 44:65-92 (1988);Verhoeyen et al., Science 239:1534-1536 (1988); Padlan, Molec. Immun.31(3):169-217 (1994); Kashmiri et al., Methods 36:25-34 (2005)(describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498(1991) (describing “resurfacing”); Dall'Acqua et al., Methods 36:43-60(2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61-68(2005) and Klimka et al., Br. J. Cancer 83:252-260 (2000) (describingthe “guided selection” approach to FR shuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

4. Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HUMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

5. Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics. Such methods are reviewed, e.g., inHoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien etal., ed., Human Press, Totowa, N.J., 2001) and further described, e.g.,in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992);Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo,ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472(2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas.

Alternatively, the naive repertoire can be cloned (e.g., from human) toprovide a single source of antibodies to a wide range of non-self andalso self antigens without any immunization as described by Griffiths etal., EMBO J, 12: 725-734 (1993). Finally, naive libraries can also bemade synthetically by cloning unrearranged V-gene segments from stemcells, and using PCR primers containing random sequence to encode thehighly variable CDR3 regions and to accomplish rearrangement in vitro,as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388(1992). Patent publications describing human antibody phage librariesinclude, for example: U.S. Pat. No. 5,750,373, and US Patent PublicationNos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126,2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

6. Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for FAP and the other is for any other antigen. Incertain embodiments, bispecific antibodies may bind to two differentepitopes of FAP. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express FAP. Bispecific antibodies canbe prepared as full length antibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983), WO 93/08829, and Traunecker et al., EMBOJ. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g., U.S.Pat. No. 5,731,168).

Multi-specific antibodies may also be made by engineering electrostaticsteering effects for making antibody Fc-heterodimeric molecules (WO2009/089004A1); cross-linking two or more antibodies or fragments (see,e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science 229:81(1985)); using leucine zippers to produce bi-specific antibodies (see,e.g., Kostelny et al., J. Immunol. 148(5):1547-1553 (1992)); using“diabody” technology for making bispecific antibody fragments (see,e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448(1993)); and using single-chain Fv (scFv) dimers (see,e.g. Gruber etal., J. Immunol., 152:5368 (1994)); and preparing trispecific antibodiesas described, e.g., in Tutt et al. J. Immunol. 147: 60 (1991).Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to FAP as well asanother, different antigen (see, US 2008/0069820, for example).

7. Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

a) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Amino acid substitutions canresult in replacing one amino acid with another amino acid havingsimilar structural and/or chemical properties, e.g., conservative aminoacid replacements.

“Conservative” amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, phenylalanine, tryptophan, and methionine;polar neutral amino acids include serine, threonine, cysteine, tyrosine,asparagine, and glutamine; positively charged (basic) amino acidsinclude arginine, lysine, and histidine; and negatively charged (acidic)amino acids include aspartic acid and glutamic acid. Conservativesubstitutions are shown in Table 2 under the heading of “preferredsubstitutions.” More substantial changes are provided in Table 2 underthe heading of “exemplary substitutions,” and as further described belowin reference to amino acid side chain classes. Amino acid substitutionsmay be introduced into an antibody of interest and the products screenedfor a desired activity, e.g., retained/improved antigen binding,decreased immunogenicity, or improved ADCC or CDC.

TABLE 2 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine;Ile; Val; Met; Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. For example, amino acid substitutionscan also result in replacing one amino acid with another amino acidhaving different structural and/or chemical properties, for example,replacing an amino acid from one group (e.g., polar) with another aminoacid from a different group (e.g., basic). The variation allowed may beexperimentally determined by systematically making insertions,deletions, or substitutions of amino acids in a polypeptide moleculeusing recombinant DNA techniques and assaying the resulting recombinantvariants for activity.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) Insome embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized. HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as Arg, Asp, His, Lys, and Glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, it may be beneficial to analyze a crystal structure of anantigen-antibody complex to identify contact points between the antibodyand antigen. Such contact residues and neighboring residues may betargeted or eliminated as candidates for substitution. Variants may bescreened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g. for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

b) Glycosylation Variants

In some embodiments, modifications of the oligosaccharide in an antibodyof the invention may be made in order to create antibody variants withcertain improved properties.

In one aspect, the present invention provides glycoforms of anti-FAPantibodies having increased effector function, includingantibody-dependent cellular cytotoxicity. Glycosylation engineering ofantibodies has been previously described. See, e.g., U.S. Pat. No.6,602,684, incorporated herein by reference in its entirety. Methods ofproducing anti-FAP antibodies from host cells that have altered activityof genes involved in glyocsylation are also described herein in detail(see, e.g, section entitled “Recombinant Methods and Compositions”below).

An IgG molecule carries two N-linked oligosaccharides in its Fc region,one on each heavy chain. As any glycoprotein, an antibody is produced asa population of glycoforms which share the same polypeptide backbone buthave different oligosaccharides attached to the glycosylation sites. Theoligosaccharides normally found in the Fc region of serum IgG are ofcomplex bi-antennary type (Wormald et al., Biochemistry 36:130-38(1997), with a low level of terminal sialic acid and bisectingN-acetylglucosamine (GlcNAc), and a variable degree of terminalgalactosylation and core fucosylation (fucose attached to a GlcNAcresidue in the “stem” of the biantennary oligosaccharide structure).Some studies suggest that the minimal carbohydrate structure requiredfor FcγR binding lies within the oligosaccharide core. Lund et al., J.Immunol. 157:4963-69 (1996).

The mouse- or hamster-derived cell lines used in industry and academiafor production of antibodies normally attach the requiredoligosaccharide determinants to Fc sites. IgGs expressed in these celllines lack, however, the bisecting GlcNAc found in low amounts in serumIgGs. Lifely et al., Glycobiology 318:813-22 (1995). In the N-linkedglycosylation pathway, a bisecting GlcNAc is added by GnTIII. Schachter,Biochem. Cell Biol. 64:163-81 (1986).

Umaña et al. used a single, antibody-producing CHO cell line that waspreviously engineered to express, in an externally-regulated fashion,different levels of a cloned GnTIII enzyme gene (Umaña, P., et al.,Nature Biotechnol. 17:176-180 (1999)). This approach established for thefirst time a rigorous correlation between expression of aglycosyltransferase (e.g., GnTIII) and the ADCC activity of the modifiedantibody. Thus, the invention contemplates anti-FAP antibodies,comprising an Fc region or region equivalent to an Fc region havingaltered glycosylation resulting from changing the expression level of aglycosyltransferase gene in the antibody-producing host cell. In aspecific embodiment, the change in gene expression level is an increasein GnTIII activity. Increased GnTIII activity results in an increase inthe percentage of bisected oligosaccharides, as well as a decrease inthe percentage of fucosylated oligosaccharides, in the Fc region of theantibody. This antibody, or fragment thereof, has increased Fc receptorbinding affinity and increased effector function.

Antibodies are provided with bisected oligosaccharides, e.g., in which abiantennary oligosaccharide attached to the Fc region of the antibody isbisected by GlcNAc. Such antibody variants may have reduced fucosylationand/or improved ADCC function. Examples of such antibody variants aredescribed, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No.6,602,684 (Umaña et al.); and US 2005/0123546 (Umaña et al.).

In one embodiment, the anti-FAP antibodies of the invention have anincreased proportion of bisected oligosaccharides in the Fc region as aresult of the modification of their oligosaccharides by the methods ofthe present invention. In one embodiment, the percentage of bisectedN-linked oligosaccharides in the Fc region of the anti-FAP antibodies ofthe invention is at least about 10% to about 100%, specifically at leastabout 50%, more specifically, at least about 60%, at least about 70%, atleast about 80%, or at least about 90-95% of the total oligosaccharides.The bisected oligosaccharides may be of the hybrid or complex type.

In another embodiment, the anti-FAP antibodies of the invention have anincreased proportion of nonfucosylated oligosaccharides in the Fc regionas a result of the modification of their oligosaccharides by the methodsof the present invention. In one embodiment, the percentage ofnonfucosylated oligosaccharides is at least about 20% to about 100%,specifically at least about 50%, at least about 60% to about 70%, andmore specifically, at least about 75%. The nonfucosylatedoligosaccharides may be of the hybrid or complex type.

The amount of fucose is determined by calculating the average amount offucose within the sugar chain at Asn297, relative to the sum of allglycostructures attached to Asn 297 (e. g. complex, hybrid and highmannose structures) as measured by MALDI-TOF mass spectrometry, asdescribed for example in WO 2008/077546. Asn297 refers to the asparagineresidue located at about position 297 in the Fc region (EU numbering ofFc region residues); however, Asn297 may also be located about ±3 aminoacids upstream or downstream of position 297, i.e., between positions294 and 300, due to minor sequence variations in antibodies. Therelative amount of fucose is the percentage of fucose-containingstructures related to all glycostructures identified in an N-GlycosidaseF treated sample (e. g. complex, hybrid and high mannose structures) byMALDI-TOF MS. Such fucosylation variants may have improved ADCCfunction.

The glycoengineering methodology that can be used with the anti-FAPantibodies of the present invention has been described in greater detailin U.S. Pat. No. 6,602,684, U.S. Pat. Appl. Publ. No. 2004/0241817 A1,U.S. Pat. Appl. Publ. No. 2003/0175884 A1, Provisional U.S. PatentApplication No. 60/441,307 and WO 2004/065540, the entire contents ofeach of which is incorporated herein by reference in its entirety. Theanti-FAP antibodies of the present invention can alternatively beglycoengineered to have reduced fucose residues in the Fc regionaccording to the techniques disclosed in U.S. Pat. Appl. Pub. No.2003/0157108 (Genentech), or in EP 1 176 195 A1, WO 03/084570, WO03/085119 and U.S. Pat. Appl. Pub. Nos. 2003/0115614, 2004/093621,2004/110282, 2004/110704, 2004/132140, Niwa et al., J Immunol Methods306, 151/160 (2006), U.S. Pat. No. 6,946,292 (Kyowa). Glycoengineeredanti-FAP antibodies of the invention may also be produced in expressionsystems that produce modified glycoproteins, such as those taught inU.S. Pat. Appl. Pub. No. 60/344,169 and WO 03/056914 (GlycoFi, Inc.) orin WO 2004/057002 and WO 2004/024927 (Greenovation).

Further examples of publications related to “defucosylated” or“fucose-deficient” antibody variants include: WO 2000/61739; WO2001/29246; US 2002/0164328; US 2004/0109865; WO 2005/035586; WO2005/035778; W02005/053742; W02002/031140; Okazaki et al. J. Mol. Biol.336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614(2004). Examples of cell lines capable of producing defucosylatedantibodies include Lec13 CHO cells deficient in protein fucosylation(Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl NoUS 2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,especially at Example 11), and knockout cell lines, such asalpha-l,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g.,Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al.,Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

In a particular embodiment, the anti-FAP antibodies of the inventionhave an increased proportion of bisected, nonfucosylatedoligosaccharides in the Fc region. The bisected, nonfucosylatedoligosaccharides may be either hybrid or complex. Specifically, themethods of the present invention may be used to produce anti-FAPantibodies in which at least about 10% to about 100%, specifically atleast about 15%, more specifically at least about 20% to about 25%, andmore specifically at least about 30% to about 35% of theoligosaccharides in the Fc region of the antigen binding molecule arebisected, nonfucosylated. The anti-FAP antibodies of the presentinvention may also comprise an Fc region in which at least about 10% toabout 100%, specifically at least about 15%, more specifically at leastabout 20% to about 25%, and more specifically at least about 30% toabout 35% of the oligosaccharides in the Fc region of the antibody arebisected hybrid nonfucosylated.

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Antibody variants with at least one galactose residue in theoligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

Increases in ADCC or other effector functions of the anti-FAP antibodiesof the present invention can also achieved by increasing affinity of theantigen binding molecule for FAP, for example by affinity maturation orother methods of improving affinity (see Tang et al., J. Immunol. 2007,179:2815-2823), or by amino acid modifications in the Fc region asdescribed below. Combinations of these approaches are also encompassedby the present invention.

c) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive methods may beemployed (see, for example, ACTI™ non-radioactive cytotoxicity assay forflow cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.Proc. Nat'l Acad. Sci. USA 95:652-656 (1998).

C1q binding assays may also be carried out to confirm that the antibodyis unable to bind C1q and hence lacks CDC activity. See, e.g., C1q andC3c binding ELISA in WO 2006/029879 and WO 2005/100402. To assesscomplement activation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues).

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.). Those antibodies comprise an Fc regionwith one or more substitutions therein which improve binding of the Fcregion to FcRn. Such Fc variants include those with substitutions at oneor more of Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307,311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434,e.g., substitution of Fc region residue 434 (U.S. Pat. No. 7,371,826).

For further examples concerning Fc region variants see also U.S. Pat.Appl. Nos. 60/439,498; 60/456,041; 60/514,549; or WO 2004/063351(variant Fc regions with increased binding affinity due to amino acidmodification); or U.S. patent application Ser. No. 10/672,280 or WO2004/099249 (Fc variants with altered binding to FcγR due to amino acidmodification), Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351.

d) Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an antibody conjugate, asdescribed further herein. In certain embodiments, any one or more of thefollowing residues may be substituted with cysteine: V205 (Kabatnumbering) of the light chain; A118 (EU numbering) of the heavy chain;and S400 (EU numbering) of the heavy chain Fc region. Cysteineengineered antibodies may be generated as described, e.g., in U.S. Pat.No. 7,521,541.

e) Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylatedpolyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.Polyethylene glycol propionaldehyde may have advantages in manufacturingdue to its stability in water. The polymer may be of any molecularweight, and may be branched or unbranched. The number of polymersattached to the antibody may vary, and if more than one polymer areattached, they can be the same or different molecules. In general, thenumber and/or type of polymers used for derivatization can be determinedbased on considerations including, but not limited to, the particularproperties or functions of the antibody to be improved, whether theantibody derivative will be used in a therapy under defined conditions,etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

B. Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated polynucleotide encoding an anti-FAP antibody described hereinis provided. Such polynucleotide may encode an amino acid sequencecomprising the VL and/or an amino acid sequence comprising the VH of theantibody (e.g., the light and/or heavy chains of the antibody). In afurther embodiment, one or more vectors (e.g., cloning vectors orexpression vectors) comprising such polynucleotide are provided. In afurther embodiment, a host cell comprising such polynucleotide or suchvector is provided. In one such embodiment, a host cell comprises (e.g.,has been transformed with): (1) a vector comprising a polynucleotidethat encodes an amino acid sequence comprising the VL of the antibodyand an amino acid sequence comprising the VH of the antibody (e.g. apolycistronic vector), or (2) a first vector comprising a polynucleotidethat encodes an amino acid sequence comprising the VL of the antibodyand a second vector comprising a polynucleotide that encodes an aminoacid sequence comprising the VH of the antibody. In one embodiment, thehost cell is a eukaryotic cell, particularly a mammalian cell, e.g. aChinese Hamster Ovary (CHO), a baby hamster kidney (BHK) cell orlymphoid cell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method ofmaking an anti-FAP antibody is provided, wherein the method comprisesculturing a host cell comprising a polynucleotide encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium).

For recombinant production of an anti-FAP antibody, one or morepolynucleotide(s) encoding an antibody, e.g., as described above, areisolated and inserted into one or more vectors for further cloningand/or expression in a host cell. Methods which are well known to thoseskilled in the art can be used to construct expression vectorscontaining the coding sequence of an anti-FAP antibody along withappropriate transcriptional/translational control signals. These methodsinclude in vitro recombinant DNA techniques, synthetic techniques and invivo recombination/genetic recombination. See, for example, thetechniques described in Maniatis et al., MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Harbor Laboratory, N.Y. (1989) and Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates andWiley Interscience, N.Y (1989).

In one embodiment, one or several polynucleotides encoding an anti-FAPantibody may be expressed under the control of a constitutive promoteror, alternatively, a regulated expression system. Suitable regulatedexpression systems include, but are not limited to, atetracycline-regulated expression system, an ecdysone-inducibleexpression system, a lac-switch expression system, aglucocorticoid-inducible expression system, a temperature-induciblepromoter system, and a metallothionein metal-inducible expressionsystem. If several different polynucleotides encoding an antibody of thepresent invention are comprised within the host cell system, some ofthem may be expressed under the control of a constitutive promoter,while others are expressed under the control of a regulated promoter.

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.,2003), pp. 245-254, describing expression of antibody fragments in E.coli.) After expression, the antibody may be isolated from the bacterialcell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006). Such expression systems are alsotaught in U.S. Pat. Appl. No. 60/344,169 and WO 03/056914 (methods forproducing human-like glycoprotein in a non-human eukaryotic host cell).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells. Plant cell cultures can also be utilized ashosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548,7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology forproducing antibodies in transgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293Tcells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

Stable expression is generally preferred to transient expression becauseit typically achieves more reproducible results and also is moreamenable to large-scale production; however, it is within the skill ofone in the art to determine whether transient expression is better for aparticular situation.

The present invention is further directed to a method for modifying theglycosylation profile of the anti-FAP antibodies of the presentinvention that are produced by a host cell, comprising expressing insaid host cell one or more polynucleotide(s) encoding an anti-FAPantibody and one or more polynucleotide(s) encoding a polypeptide with aglycosyltransferase activity, or a vector comprising suchpolynucleotides. Generally, any type of cultured cell line, includingthe cell lines discussed above, can be used to generate cell lines forthe production of anti-FAP antibodies with altered glycosylationpattern. Preferred cell lines include CHO cells, BHK cells, NS0 cells,SP2/0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells,PER.C6 cells or hybridoma cells, and other mammalian cells. Polypeptideswith glycosyltransferase activity includeβ(1,4)-N-acetylglucosaminyltransferase III (GnTIII), α-mannosidase II(ManII), β(1,4)-galactosyltransferase (GalT),β(1,2)-N-acetylglucosaminyltransferase I (GnTI), andβ(1,2)-N-acetylglucosaminyltransferase II (GnTII). In one embodiment, acombination of polynucleotides encoding for polynucleotides withglycosyltransferase activity are expressed in the host cell (e.g.,GnTIII and Man II). Likewise, the method also encompasses expression ofone or more polynucleotide(s) encoding the anti-FAP antibody in a hostcell in which a glycosyltransferase gene has been disrupted or otherwisedeactivated (e.g., a host cell in which the activity of the geneencoding α1,6 core fucosyltransferase has been knocked out). In aparticular embodiment, the anti-FAP antibodies of the present inventioncan be produced in a host cell that further expresses a polynucleotideencoding a polypeptide having GnTIII activity to modify theglycosylation pattern of said antibodies. In a specific embodiment, thepolypeptide having GnTIII activity is a fusion polypeptide comprisingthe Golgi localization domain of a Golgi resident polypeptide. Inanother particular embodiment, the expression of the anti-FAP antibodyof the present invention in a host cell that expresses a polynucleotideencoding a polypeptide having GnTIII activity results in anti-FAPantibodies with increased Fc receptor binding affinity and/or increasedeffector function. Accordingly, in one embodiment, the present inventionis directed to a host cell comprising (a) one or more isolatedpolynucleotide(s) comprising a sequence encoding a polypeptide havingGnTIII activity; and (b) one or more isolated polynucleotide(s) encodingan anti-FAP antibody of the present invention. In a particularembodiment, the polypeptide having GnTIII activity is a fusionpolypeptide comprising the catalytic domain of GnTIII and the Golgilocalization domain of a heterologous Golgi resident polypeptide.Particularly, said Golgi localization domain is the Golgi localizationdomain of mannosidase II. Methods for generating such fusionpolypeptides and using them to produce antibodies with increasedeffector functions are disclosed in WO2004/065540, U.S. Provisional Pat.Appl. No. 60/495,142 and U.S. Pat. Appl. Publ. No. 2004/0241817, theentire contents of which are expressly incorporated herein by reference.In another embodiment, the host cell additionally comprises an isolatedpolynucleotide comprising a sequence encoding a polypeptide havingmannosidase II (ManII) activity. The polynucleotide(s) encodingpolypeptide(s), like the polynucleotide(s) encoding the anti-FAPantibody, may be expressed under the control of a constitutive promoteror, alternately, a regulated expression system. Such systems are wellknown in the art, and include the systems discussed above.

The host cells which contain the coding sequence of the anti-FAPantibody and/or the coding sequence of polypeptides havingglycosyltransferase activity, and which express the biologically activegene products may be identified e.g. by DNA-DNA or DNA-RNAhybridization; the presence or absence of “marker” gene functions;assessing the level of transcription as measured by the expression ofthe respective mRNA transcripts in the host cell; or detection of thegene product as measured by immunoassay or by its biologicalactivity—methods which are well known in the art. GnTIII or Man IIactivity can be detected e.g. by employing a lectin which binds tobiosynthetis products of GnTIII or ManII, respectively. An example forsuch a lectin is the E₄-PHA lectin which binds preferentially tooligosaccharides containing bisecting GlcNAc. Biosynthesis products(i.e. specific oligosaccharide structures) of polypeptides having GnTIIIor ManII activity can also be detected by mass spectrometric analysis ofoligosaccharides released from glycoproteins produced by cellsexpressing said polypeptides. Alternatively, a functional assay whichmeasures the increased Fc receptor binding or increased effectorfunction mediated by antibodies produced by the cells engineered withthe polynucleotide encoding a polypeptide having GnTIII activity may beused.

The present invention is also directed to a method for producing ananti-FAP antibody having modified oligosaccharides, comprising (a)culturing a host cell engineered to express at least one polynucleotideencoding a polypeptide having glycosyltransferase activity underconditions which permit the production of an anti-FAP antibody accordingto the present invention, wherein said polypeptide havingglycosyltransferase activity is expressed in an amount sufficient tomodify the oligosaccharides in the Fc region of said anti-FAP antibodyproduced by said host cell; and (b) isolating said anti-FAP antibody. Inone embodiment, the polypeptide having glycosyltransferase activity isGnTIII. In another embodiment, there are two polypeptides havingglycosyltransferase activity. In a particular embodiment, the twopeptides having glycosyltransferase activity are GnTIII and ManII Inanother embodiment, the polypeptide having glycosyltransferase activityis a fusion polypeptide comprising the catalytic domain of GnTIII. In amore specific embodiment, the fusion polypeptide further comprises theGolgi localization domain of a Golgi resident polypeptide. Particularly,the Golgi localization domain is the localization domain of mannosidaseII or GnTI, most particularly the localization domain of mannosidase II.Alternatively, the Golgi localization domain is selected from the groupconsisting of: the localization domain of mannosidase I, thelocalization domain of GnTII, and the localization domain of α1,6 corefucosyltransferase.

In a particular embodiment, the modified anti-FAP antibody produced bythe host cell or method described above has an IgG constant region or afragment thereof comprising the Fc region. In another particularembodiment the anti-FAP antibody is a humanized or human antibody or afragment thereof comprising an Fc region.

The anti-FAP antibody with altered glycosylation produced by the hostcell or method described above typically exhibit increased Fc receptorbinding affinity and/or increased effector function as a result of themodification of the host cell (e.g., by expression of aglycosyltransferase gene). Preferably, the increased Fc receptor bindingaffinity is increased binding to an activating Fcγ receptor, mostpreferably the FcγRIIIa receptor. The increased effector function ispreferably an increase in one or more of the following: increasedantibody-dependent cellular cytotoxicity, increased antibody-dependentcellular phagocytosis (ADCP), increased cytokine secretion, increasedimmune-complex-mediated antigen uptake by antigen-presenting cells,increased Fc-mediated cellular cytotoxicity, increased binding to NKcells, increased binding to macrophages, increased binding topolymorphonuclear cells (PMNCs), increased binding to monocytes,increased crosslinking of target-bound antibodies, increased directsignaling inducing apoptosis, increased dendritic cell maturation, andincreased T cell priming.

C. Assays

Anti-FAP antibodies provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

1. Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc.

In another aspect, competition assays may be used to identify anantibody that competes with another specific anti-FAP antibody forbinding to FAP. In certain embodiments, such a competing antibody bindsto the same epitope (e.g., a linear or a conformational epitope) that isbound by said other specific anti-FAP antibody. Detailed exemplarymethods for mapping an epitope to which an antibody binds are providedin Morris (1996) “Epitope Mapping Protocols,” in Methods in MolecularBiology vol. 66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized FAP is incubated in asolution comprising a first labeled antibody that binds to FAP (e.g. the3F2 antibody described in the Examples) and a second unlabeled antibodythat is being tested for its ability to compete with the first antibodyfor binding to FAP. The second antibody may be present in a hybridomasupernatant. As a control, immobilized FAP is incubated in a solutioncomprising the first labeled antibody but not the second unlabeledantibody. After incubation under conditions permissive for binding ofthe first antibody to FAP, excess unbound antibody is removed, and theamount of label associated with immobilized FAP is measured. If theamount of label associated with immobilized FAP is substantially reducedin the test sample relative to the control sample, then that indicatesthat the second antibody is competing with the first antibody forbinding to FAP. See Harlow and Lane (1988) Antibodies: A LaboratoryManual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

2. Activity Assays

In one aspect, assays are provided for identifying anti-FAP antibodiesthereof having biological activity. Biological activity may include,e.g., lysis of targeted cells, antibody-dependent cell-mediatedcytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), orinduction of apoptosis. Antibodies having such biological activity invivo and/or in vitro are also provided. In certain embodiments, anantibody of the invention is tested for such biological activity.Exemplary assays for testing ADCC are described hereinbefore (see under“Definitions”: “antibody having increased ADCC”) and in Example 11.Assays for detecting cell lysis (e.g. by measurement of LDH release) orapoptosis (e.g. using the TUNEL assay) are well known in the art. Assaysfor measuring ADCC or CDC are also described in WO 2004/065540 (seeExample 1 therein), the entire content of which is incorporated hereinby reference.

D. Antibody Conjugates

The invention also provides conjugates comprising an anti-FAP antibodyherein conjugated to one or more cytotoxic agents, such aschemotherapeutic agents or drugs, growth inhibitory agents, toxins(e.g., protein toxins, enzymatically active toxins of bacterial, fungal,plant, or animal origin, or fragments thereof), or radioactive isotopes.

In one embodiment, in an antibody-drug conjugate (ADC) an antibody isconjugated to one or more drugs, including but not limited to amaytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064 and EuropeanPatent EP 0 425 235 B1); an auristatin such as monomethylauristatin drugmoieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivativethereof (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285,5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al., CancerRes. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928(1998)); an anthracycline such as daunomycin or doxorubicin (see Kratzet al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic& Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem.16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834(2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532(2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat.No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel,paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; andCC1065.

In another embodiment, an antibody conjugate comprises an antibody asdescribed herein conjugated to an enzymatically active toxin or fragmentthereof, including but not limited to diphtheria A chain, nonbindingactive fragments of diphtheria toxin, exotoxin A chain (from Pseudomonasaeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

In another embodiment, an antibody conjugate comprises an antibody asdescribed herein conjugated to a radioactive atom to form aradioconjugate. A variety of radioactive isotopes are available for theproduction of radioconjugates. Examples include At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu.When the radioconjugate is used for detection, it may comprise aradioactive atom for scintigraphic studies, for example tc99m or I123,or a spin label for nuclear magnetic resonance (NMR) imaging (also knownas magnetic resonance imaging, mri), such as iodine-123 again,iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17,gadolinium, manganese or iron.

Conjugates of an antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio)propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of a cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Res. 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The antibody conjugates herein expressly contemplate, but are notlimited to such conjugates prepared with cross-linker reagentsincluding, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS,MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS,sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate) which are commercially available(e.g., from Pierce Biotechnology, Inc., Rockford, Ill., U.S.A).

E. Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-FAP antibodies provided hereinis useful for detecting the presence of FAP in a biological sample. Theterm “detecting” as used herein encompasses quantitative or qualitativedetection. In certain embodiments, a biological sample comprises a cellor tissue, such as cells or tissues from brain, breast, colon, kidney,liver, lung, ovary, pancreas, prostate, skeletal muscle, skin, smallintestine, stomach or uterus, including also cells or tissues tumors ofthese organs.

In one embodiment, an anti-FAP antibody for use in a method of diagnosisor detection is provided. In a further aspect, a method of detecting thepresence of FAP in a biological sample is provided. In certainembodiments, the method comprises contacting the biological sample,optionally with a control sample, with an anti-FAP antibody as describedherein under conditions permissive for binding of the anti-FAP antibodyto FAP, and detecting whether a complex is formed between the anti-FAPantibody and FAP. Such method may be an in vitro or in vivo method. Inone embodiment, an anti-FAP antibody is used to select subjects eligiblefor therapy with an anti-FAP antibody, e.g. where FAP is a biomarker forselection of patients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include disorders associated with the expression of FAP, suchas cancer and certain inflammatory conditions. In one aspect, a methodof diagnosing disease in a subject is provided, said method comprisingadministering to said subject an effective amount of a diagnostic agent,wherein said diagnostic agent comprises an anti-FAP antibody asdescribed herein and a label, typically an imaging agent, that allowsdetection of a complex of said diagnostic agent and FAP.

In certain embodiments, labeled anti-FAP antibodies are provided. Labelsinclude, but are not limited to, labels or moieties that are detecteddirectly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

F. Pharmaceutical Formulations

Pharmaceutical formulations of an anti-FAP antibody as described hereinare prepared by mixing such antibody having the desired degree of puritywith one or more optional pharmaceutically acceptable carriers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions.Pharmaceutically acceptable carriers are generally nontoxic torecipients at the dosages and concentrations employed, and include, butare not limited to: buffers such as phosphate, citrate, acetate andother organic acids; antioxidants including ascorbic acid andmethionine; preservatives (such as octadecyldimethylbenzyl ammoniumchloride; hexamethonium chloride; benzalkonium chloride; benzethoniumchloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methylor propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; andm-cresol); low molecular weight (less than about 10 residues)polypeptides; proteins, such as serum albumin, gelatin, orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, histidine, arginine,or lysine; monosaccharides, disaccharides, and other carbohydratesincluding glucose, mannose, or dextrins; chelating agents such as EDTA;sugars such as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude insterstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958.

Aqueous antibody formulations include those described in U.S. Pat. No.6,171,586 and WO2006/044908, the latter formulations including ahistidine-acetate buffer.

The formulation herein may also contain more than one active ingredientsas necessary for the particular indication being treated, preferablythose with complementary activities that do not adversely affect eachother. For example, if the disease to be treated is cancer, it may bedesirable to further provide one or more anti-cancer agents, e.g. achemotherapeutic agent, an inhibitor of tumor cell proliferation, or anactivator of tumor cell apoptosis. Such active ingredients are suitablypresent in combination in amounts that are effective for the purposeintended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. The formulationsto be used for in vivo administration are generally sterile. Sterilitymay be readily accomplished, e.g., by filtration through sterilefiltration membranes.

The molecules described herein may be in a variety of dosage forms whichinclude, but are not limited to, liquid solutions or suspensions,tablets, pills, powders, suppositories, polymeric microcapsules ormicrovesicles, liposomes, and injectable or infusible solutions. Thepreferred form depends upon the mode of administration and thetherapeutic application, but will typically be injectable or infusiblesolutions.

G. Therapeutic Methods and Compositions

Any of the anti-FAP antibodies or pharmaceutical formulations comprisingthe anti-FAP antibodies provided herein may be used in therapeuticmethods.

The anti-FAP antibodies provided herein can be used for treatingdiseases characterized by FAP expression, particularly by abnormalexpression (e.g. overexpression, or expression in a different pattern inthe cell) of FAP compared to normal tissue of the same cell type. FAP isabnormally expressed (e.g. overexpressed) in many human tumors comparedto non-tumor tissue of the same cell type. Thus, the anti-FAP antibodiesprovided herein are particularly useful in the prevention of tumorformation, eradication of tumors and inhibition of tumor growth ormetastasis. The anti-FAP antibodies provided herein can be used to treatany tumor expressing FAP. Particular malignancies that can be treated bythe anti-FAP antibodies provided herein include, for example, lungcancer, colon cancer, gastric cancer, breast cancer, head and neckcancer, skin cancer, liver cancer, kidney cancer, prostate cancer,pancreatic cancer, brain cancer, cancer of the skeletal muscle.

The anti-FAP antibodies disclosed herein can be used to inhibit tumorgrowth or kill tumor cells. For example, the anti-FAP antibodies canbind to FAP that is on the membrane or cell surface of cancerous cells(tumor cells or cells of the tumor stroma) and elicit, e.g., ADCC orother effector mediated killing of the cancerous cells.

The anti-FAP antibodies can alternatively be used in order to block thefunction of FAP, particularly by physically interfering with its bindingof another compound. For example, the antigen binding molecules can beused to block the enzymatic activity of FAP (e.g. serine peptidase,gelatinase, collagenase activity), FAP mediated ECM degradation, and/orFAP mediated cell invasion or migration.

In one aspect, an anti-FAP antibody for use as a medicament is provided.In further aspects, an anti-FAP antibody for use in treating a diseasecharacterized by expression of FAP is provided. In certain embodiments,an anti-FAP antibody for use in a method of treatment is provided. Incertain embodiments, the invention provides an anti-FAP antibody for usein a method of treating an individual having a disease characterized byexpression of FAP, comprising administering to the individual aneffective amount of the anti-FAP antibody. In one such embodiment, themethod further comprises administering to the individual an effectiveamount of at least one additional therapeutic agent, e.g., as describedbelow. In further embodiments, the invention provides an anti-FAPantibody for use in inducing lysis of a cell. In certain embodiments,the invention provides an anti-FAP antibody for use in a method ofinducing lysis of a cell in an individual comprising administering tothe individual an effective amount of the anti FAP antibody to inducelysis of a cell. An “individual” according to any of the aboveembodiments is preferably a human. A “disease characterized byexpression of FAP” according to any of the above embodiments ispreferably cancer, most preferably a cancer selected from the group oflung cancer, colon cancer, gastric cancer, breast cancer, head and neckcancer, skin cancer, liver cancer, kidney cancer, prostate cancer,pancreatic cancer, brain cancer, cancer of the skeletal muscle. A “cell”according to any of the above embodiments is preferably a cell presentin a tumor, such as a tumor cell or a cell of the tumor stroma, mostpreferably a tumor cell. “FAP expression” according to any of the aboveembodiments preferably is abnormal expression, such as overexpression orexpression in a different pattern in the cell, compared to normal tissueof the same cell type.

In a further aspect, the invention provides for the use of an anti-FAPantibody in the manufacture or preparation of a medicament. In oneembodiment, the medicament is for treatment of a disease characterizedby expression of FAP. In a further embodiment, the medicament is for usein a method of treating a disease characterized by expression of FAPcomprising administering to an individual having a disease characterizedby expression of FAP an effective amount of the medicament. In one suchembodiment, the method further comprises administering to the individualan effective amount of at least one additional therapeutic agent, e.g.,as described below. In a further embodiment, the medicament is forinducing lysis of a cell. In a further embodiment, the medicament is foruse in a method of inducing lysis of a cell in an individual comprisingadministering to the individual an amount effective of the medicament toinducing lysis of a cell. An “individual” according to any of the aboveembodiments is preferably a human. A “disease characterized byexpression of FAP” according to any of the above embodiments ispreferably cancer, most preferably a cancer selected from the group oflung cancer, colon cancer, gastric cancer, breast cancer, head and neckcancer, skin cancer, liver cancer, kidney cancer, prostate cancer,pancreatic cancer, brain cancer, cancer of the skeletal muscle. A “cell”according to any of the above embodiments is preferably a cell presentin a tumor, such as a tumor cell or a cell of the tumor stroma, mostpreferably a tumor cell. “FAP expression” according to any of the aboveembodiments preferably is abnormal expression, such as overexpression orexpression in a different pattern in the cell, compared to normal tissueof the same cell type.

In a further aspect, the invention provides a method for treating adisease characterized by expression of FAP. In one embodiment, themethod comprises administering to an individual having such diseasecharacterized by expression of FAP an effective amount of an anti-FAPantibody. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent, as described below. In a furtherembodiment, the invention provides a method for inducing lysis of a cellin an individual. In one embodiment, the method comprises administeringto the individual an effective amount of an anti-FAP antibody to inducelysis of a cell. An “individual” according to any of the aboveembodiments may be a human. A “disease characterized by expression ofFAP” according to any of the above embodiments is preferably cancer,most preferably a cancer selected from the group of lung cancer, coloncancer, gastric cancer, breast cancer, head and neck cancer, skincancer, liver cancer, kidney cancer, prostate cancer, pancreatic cancer,brain cancer, cancer of the skeletal muscle. A “cell” according to anyof the above embodiments is preferably a cell present in a tumor, suchas a tumor cell or a cell of the tumor stroma, most preferably a tumorcell. “FAP expression” according to any of the above embodimentspreferably is abnormal expression, such as overexpression or expressionin a different pattern in the cell, compared to normal tissue of thesame cell type.

In a further aspect, the invention provides pharmaceutical formulationscomprising any of the anti-FAP antibodies provided herein, e.g., for usein any of the above therapeutic methods. In one embodiment, apharmaceutical formulation comprises any of the anti-FAP antibodiesprovided herein and one or more pharmaceutically acceptable carrier. Inanother embodiment, a pharmaceutical formulation comprises any of theanti-FAP antibodies provided herein and at least one additionaltherapeutic agent, e.g., as described below.

Antibodies of the invention can be used either alone or in combinationwith other agents in a therapy. For instance, an antibody of theinvention may be co-administered with at least one additionaltherapeutic agent. In certain embodiments, an additional therapeuticagent is an anti- cancer agent, e.g. a chemotherapeutic agent, aninhibitor of tumor cell proliferation, or an activator of tumor cellapoptosis.

Such combination therapies noted above encompass combined administration(where two or more therapeutic agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,simultaneously, and/or following, administration of the additionaltherapeutic agent and/or adjuvant. Antibodies of the invention can alsobe used in combination with radiation therapy. An antibody of theinvention (and any additional therapeutic agent) can be administered byany suitable means, including parenteral, intrapulmonary, andintranasal, and, if desired for local treatment, intralesionaladministration. Parenteral administration includes intramuscular,intravenous, intraarterial, intraperitoneal, or subcutaneousadministration. Intravenous administration is typically preferred.However, the intraperitoneal route is expected to be particularlyuseful, for example, in the treatment of colorectal tumors. Dosing canbe by any suitable route, e.g. by injections, such as intravenous orsubcutaneous injections, depending in part on whether the administrationis brief or chronic. Various dosing schedules including but not limitedto single or multiple administrations over various time-points, bolusadministration, and pulse infusion are contemplated herein.

Antibodies of the invention would be formulated, dosed, and administeredin a fashion consistent with good medical practice. Factors forconsideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount of antibodypresent in the formulation, the type of disorder or treatment, and otherfactors discussed above. These are generally used in the same dosagesand with administration routes as described herein, or about from 1 to99% of the dosages described herein, or in any dosage and by any routethat is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with one ormore other additional therapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment would generally be sustaineduntil a desired suppression of disease symptoms occurs. One exemplarydosage of the antibody would be in the range from about 0.05 mg/kg toabout 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg,4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administeredto the patient. Such doses may be administered intermittently, e.g.every week or every three weeks (e.g. such that the patient receivesfrom about two to about twenty, or e.g. about six doses of theantibody). An initial higher loading dose, followed by one or more lowerdoses may be administered. However, other dosage regimens may be useful.The progress of this therapy is easily monitored by conventionaltechniques and assays.

It is understood that any of the above formulations or therapeuticmethods may be carried out using an antibody conjugate of the inventionin place of or in addition to an anti-FAP antibody.

H. Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, IV solution bags, etc. The containers may be formedfrom a variety of materials such as glass or plastic. The containerholds a composition which is by itself or combined with anothercomposition effective for treating, preventing and/or diagnosing thecondition and may have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle). At least one active agent in thecomposition is an antibody of the invention. The label or package insertindicates that the composition is used for treating the condition ofchoice. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic or otherwise therapeutic agent. The article ofmanufacture in this embodiment of the invention may further comprise apackage insert indicating that the compositions can be used to treat aparticular condition. Alternatively, or additionally, the article ofmanufacture may further comprise a second (or third) containercomprising a pharmaceutically-acceptable buffer, such as bacteriostaticwater for injection (BWFI), phosphate-buffered saline, Ringer's solutionand dextrose solution. It may further include other materials desirablefrom a commercial and user standpoint, including other buffers,diluents, filters, needles, and syringes.

It is understood that any of the above articles of manufacture mayinclude an antibody conjugate of the invention in place of or inaddition to an anti-FAP antibody.

III. EXAMPLES

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

Example 1

Recombinant DNA Techniques

Standard methods were used to manipulate DNA as described in Sambrook,J. et al., Molecular cloning: A laboratory manual; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. The molecularbiological reagents were used according to the manufacturer'sinstructions. DNA sequences were determined by double strand sequencing.In some cases desired gene segments were prepared by Geneart AG(Regensburg, Germany) from synthetic oligonucleotides and PCR productsby automated gene synthesis. The gene segments which are flanked bysingular restriction endonuclease cleavage sites were cloned into pGA18(ampR) plasmids. The plasmid DNA was purified from transformed bacteriaand concentration determined by UV spectroscopy. The DNA sequence of thesubcloned gene fragments was confirmed by DNA sequencing. Gene Segmentswere designed with suitable restriction sites to allow sub-cloning intothe respective expression vectors.

General information regarding the nucleotide sequences of humanimmunoglobulin light and heavy chains is given in: Kabat, E. A. et al.,(1991) Sequences of Proteins of Immunological Interest, Fifth Ed., NIHPublication No 91-3242. For expression, all constructs contained a5′-end DNA sequence coding for a leader peptide which targets proteinsfor secretion in eukaryotic cells. SEQ ID NOs 323-331 give exemplaryleader peptides and polynucleotide sequences encoding them.

Preparation of (Glycoengineered) Antibodies

The full antibody heavy and light chain DNA sequences have been obtainedby subcloning the variable regions in frame with either the constantheavy chain or the constant light chain pre-inserted into the respectiverecipient mammalian expression vector. The antibody expression wasdriven by an MPSV promoter and the vector carries a synthetic polyAsignal sequence at the 3′ end of the CDS. In addition each vectorcontains an EBV OriP sequence.

Antibodies are produced by co-transfecting HEK293-EBNA cells with themammalian antibody expression vectors using a calciumphosphate-transfection. Exponentially growing HEK293-EBNA cells aretransfected by the calcium phosphate method. Alternatively, HEK293 cellsgrowing in suspension are transfected by polyethylenimine. For theproduction of unmodified non-glycoengineered antibody, the cells aretransfected only with antibody heavy and light chain expression vectorsin a 1:1 ratio.

For the production of the glycoengineered antibody, the cells areco-transfected with two additional plasmids, one for a fusion GnTIIIpolypeptide expression (a GnT-III expression vector), and one formannosidase II expression (a Golgi mannosidase II expression vector) ata ratio of 4:4:1:1, respectively. Cells are grown as adherent monolayercultures in T flasks using DMEM culture medium supplemented with 10%FCS, and are transfected when they are between 50 and 80% confluent. Forthe transfection of a T150 flask, 15 million cells are seeded 24 hoursbefore transfection in 25 ml DMEM culture medium supplemented with FCS(at 10% V/V final), and cells are placed at 37° C. in an incubator witha 5% CO₂ atmosphere overnight. For each T150 flask to be transfected, asolution of DNA, CaCl₂ and water is prepared by mixing 94 μg totalplasmid vector DNA divided equally between the light and heavy chainexpression vectors, water to a final volume of 469 μl and 469 μl of a 1MCaCl₂ solution. To this solution, 938 μl of a 50 mM HEPES, 280 mM NaCl,1.5 mM Na₂HPO₄ solution at pH 7.05 are added, mixed immediately for 10sec and left to stand at room temperature for 20 sec. The suspension isdiluted with 10 ml of DMEM supplemented with 2% FCS, and added to theT150 in place of the existing medium. Then additional 13 ml oftransfection medium are added. The cells are incubated at 37° C., 5% CO₂for about 17 to 20 hours, then medium is replaced with 25 ml DMEM, 10%FCS. The conditioned culture medium is harvested approx. 7 dayspost-media exchange by centrifugation for 15 min at 210×g, the solutionis sterile filtered (0.22 um filter) and sodium azide in a finalconcentration of 0.01% w/v is added, and kept at 4° C.

The secreted wildtype or glycoengineered a fucosylated antibodies arepurified from cell culture supernatants by affinity chromatography usingProtein A (HiTrap ProtA, GE Healthcare) affinity chromatography.Briefly, the column was equilibrated with 20 mM sodium phosphate, 20 mMsodium citrate pH 7.5, the cell supernatant was loaded, followed by afirst wash with 20 mM sodium phosphate, 20 mM sodium citrate pH 7.5, anda second wash with 13.3 mM sodium phosphate, 20 mM sodium citrate, 500mM sodium chloride pH 5.45. The antibodies were eluted with 20 mM sodiumcitrate, 100 mM sodium chloride, 100 mM glycine pH 3. In a subsequentsize exclusion chromatographic step on a HiLoad Superdex 200 column (GEHealthcare) the buffer was exchanged to 25 mM potassium phosphate, 125mM sodium chloride, 100 mM glycine solution of pH 6.7 or alternatively140 mM sodium chloride, 20 mM histidine, pH 6.0 and the pure monomericIgG1 antibodies were collected. If required an additional cationexchange chromatography step is included between the two standardpurification steps.

The protein concentration of purified protein samples is determined bymeasuring the optical density (OD) at 280 nm, using the molar extinctioncoefficient calculated on the basis of the amino acid sequence. Purityand molecular weight of antibodies are analyzed by SDS-PAGE in thepresence and absence of a reducing agent (5 mM 1,4-dithiotreitol) andstaining with Coomassie (SimpleBlue™ SafeStain from Invitrogen). TheNuPAGE® Pre-Cast gel system (Invitrogen, USA) is used according to themanufacturer's instruction (4-20% Tris-Glycine gels or 3-12% Bis-Tris).The aggregate content of antibody samples is analyzed using a Superdex200 10/300GL analytical size-exclusion column (GE Healthcare, Sweden) in2 mM MOPS, 150 mM NaCl, 0.02% NaN₃, pH 7.3 running buffer at 25° C. Theintegrity of the amino acid backbone of reduced antibody light and heavychains is verified by NanoElectrospray Q-TOF mass spectrometry afterremoval of N-glycans by enzymatic treatment with Peptide-N Glycosidase F(Roche Molecular Biochemicals).

The results of the purification and analysis of the wild-type andglycoengineered 28H1, 29B11, 3F2 and 4G8 human IgG antibodies are shownin FIGS. 15A-D, 16A-D, 17A-D, 18A-D, 19A-D, 20A-D, 21A-D, and 22A-D. Theyields are given in the following table:

Yield [mg/L] wild- glyco- type engineered 28H1 hu IgG 46 40 29B11 hu IgG10 14 3F2 hu IgG 144 7 4G8 hu IgG 55 12.6

The oligosaccharides attached to the Fc region of the antibodies areanalysed by MALDI TOF-MS as described below. Oligosaccharides areenzymatically released from the antibodies by PNGaseF digestion. Theresulting digest solution containing the released oligosaccharides iseither prepared directly for MALDI TOF-MS analysis or is furtherdigested with EndoH glycosidase prior to sample preparation for MALDITOF-MS analysis.

Analysis of Glycostructure of (Glycoengineered) Antibodies

For determination of the relative ratios of fucose- and non-fucose(a-fucose) containing oligosaccharide structures, released glycans ofpurified antibody material are analyzed by MALDI-Tof-mass spectrometry.The antibody sample (about 50 μg) is incubated overnight at 37° C. with5 mU N-Glycosidase F (QAbio; PNGaseF: E-PNG01) in 2 mM Tris, pH 7.0, inorder to release the oligosaccharide from the protein backbone. Fordeamination of glycans acetic acid to a final concentration of 150 mM isadded and incubated for 1 h at 37° C. For analysis by MALDI TOF massspectrometry, 2 μL of the sample are mixed on the MALDI target with 2 μLDHB matrix solution (2,5-dihydroxybenzoic acid [Bruker Daltonics#201346] dissolved in 50% ethanol/5 mM NaCl at 4 mg/ml) and analysedwith MALDI TOF Mass Spectrometer Autoflex II instrument [BrukerDaltonics]. Routinely, 50-300 shots are recorded and summed up to asingle experiment. The spectra obtained are evaluated by the flexanalysis software (Bruker Daltonics) and masses are determined for theeach of the peaks detected. Subsequently, the peaks are assigned tofucose or a-fucose (non-fucose) containing carbohydrate structures bycomparing the masses calculated and the masses theoretically expectedfor the respective structures (e.g. complex, hybrid and oligo-orhigh-mannose, respectively, with and without fucose).

For determination of the ratio of hybrid structures, the antibodysamples are digested with N-Glycosidase F and Endo-Glycosidase H [QAbio;EndoH: E-EH02] concomitantly. N-Glycosidase F releases all N-linkedglycan structures (complex, hybrid and oligo- and high mannosestructures) from the protein backbone and the Endo-Glycosidase H cleavesall the hybrid type glycans additionally between the twoN-acetylglucosamine (GlcNAc) residues at the reducing end of the glycan.This digest is subsequently treated and analysed by MALDI TOF massspectrometry in the same way as described above for the N-Glycosidase Fdigested sample.

By comparing the pattern from the N-Glycosidase F digest and thecombined N-glycosidase F/Endo H digest, the degree of reduction of thesignals of a specific carbohydrate structure is used to estimate therelative content of hybrid structures. The relative amount of eachcarbohydrate structure is calculated from the ratio of the peak heightof an individual structure and the sum of the peak heights of alloligosaccharides detected. The amount of fucose is the percentage offucose-containing structures related to all carbohydrate structuresidentified in the N-Glycosidase F treated sample (e.g. complex, hybridand oligo- and high-mannose structures, resp.). The amount ofnon-fucosylation is the percentage of fucose-lacking structures relatedto all carbohydrates identified in the N-Glycosidase F treated sample(e.g. complex, hybrid and oligo- and high-mannose structures, resp.).

The degrees of non-fucosylation of the different wild-type andglycoengineered anti-FAP antibodies is given in the following table:

Non-fucosylation [%] Wild- glyco- Antibody type engineered Hu IgG 28H110 40 Hu IgG 29B11 5 27 Hu IgG 3F2(YS) 2.4 64 Hu IgG 4G8 3.8 78

Example 2

Construction of Generic Fab-Libraries

Generic antibody libraries in the Fab-format were constructed on thebasis of human germline genes using the following V-domain pairings:Vk3_(—)20 kappa light chain with VH3_(—)23 heavy chain for the DP47-3library and Vk1_(—)17 kappa light chain with VH1_(—)69 heavy chain forthe DP88-3 library. See SEQ ID NOs 1 and 2.

Both libraries were randomized in CDR3 of the light chain (L3) and CDR3of the heavy chain (H3) and were assembled from 3 fragments per libraryby splicing by overlapping extension (SOE) PCR. Fragment 1 comprises the5′ end of the antibody gene including randomized L3, fragment 2 is acentral constant fragment spanning from L3 to H3, whereas fragment 3comprises randomized H3 and the 3′ portion of the antibody gene.

The following primer combinations were used to generate libraryfragments for DP47-3 library: fragment 1 (LMB3-LibL1b_new), fragment 2(MS63-MS64), fragment 3 (Lib2H-fdseqlong). See Table 3. The followingprimer combinations were used to generate library fragments for theDP88-3 library: fragment 1 (LMB3-RJH_LIB3), fragment 2 (RJH31-RJH32) andfragment 3 (LIB88_(—)2-fdseqlong). See Table 4.

TABLE 3 SEQ ID Primers Used In the DP47-3 Library NO LMB3CAGGAAACAGCTATGACCATGATTAC 332 LibL1b_CACTTTGGTCCCCTGGCCGAACGTMNNGGGMNNM 333 new NNMNNACCCTGCTGACAGTAATACACTGCMS63 TTTCGCACAGTAATATACGGCCGTGTCC 334 MS64 ACGTTCGGCCAGGGGACCAAAGTGG 335Lib2H GGCCGTATATTACTGTGCGAAANNKNNKNNKNNK 336 NNKTTTGACTACTGGGGCCAAGGAACfdseqlong GACGTTAGTAAATGAATTTTCTGTATGAGG 337

TABLE 4 SEQ ID Primers Used in DP88-3 Library NO LMB3CAGGAAACAGCTATGACCATGATTAC 332 RJH_LIB3 GACTTTGGTGCCCTGGCCAAACGT MNN  338 GGG MNN MNN ACC MNN  CTGCAAGCAGTAATAGGTGGCAAAATC RJH31ACGTTTGGCCAGGGCACCAAAGTCGAG 339 RJH32 TCTCGCACAGTAATACACGGCGGTGTCC 340LIB88_2 GGACACCGCCGTGTATTACTGTGCGAGA- 341 [33% GAC Asp; 26% GGT Gly; 10%GAA Glu; 9% CGT Arg; 7% Lys;  6% GTT Val; 5% TCT Ser; 4% CTGLeu)1 - (23% GGT Gly; 17% TAC  Tyr; 16% TCT Ser; 11% GCT Ala;9% CGT Arg; 7% AAC Asn; 6% ACT  Thr; 6% GTT Val; 5% CCG Pro)8]-TTTGACTACTGGGGCCAAGGGACCACCGTGA CCGTCTCC fdseqlongGACGTTAGTAAATGAATTTTCTGTATGAGG 337

The PCR protocol for the production of library fragments included: 5minutes of initial denaturation at 94° C.; 25 cycles of 1 minute at 94°C., 1 minute at 58° C., and 1 minute at 72° C.; and terminal elongationfor 10 minutes at 72° C. For assembly PCR, equimolar ratios of the 3fragments were used as template. The assembly PCR protocol included: 3minutes of initial denaturation at 94° C.; and 5 cycles of 30 seconds at94° C., 1 minute at 58° C., and 2 minutes at 72° C. At this stage,primers complementary to sequence outside fragments 1-3 were added andan additional 20 cycles were performed prior to a terminal elongationfor 10 min at 72° C. After assembly of sufficient amounts of full lengthrandomized Fab constructs, the Fab constructs were digested withNcoI/NotI for the DP47-3 library and with NcoI/NheI for the DP88-3library alongside with similarly treated acceptor phagemid vector. Forthe DP47-3 library, 22.8 μg of Fab library was ligated with 16.2 μg ofphagemid vector. For the DP88-3 library, 30.6 μg of Fab library wasligated with 30.6 μg of phagemid vector.

Purified ligations were used for 68 transformations for the DP47-3library and 64 transformations for the DP88-3 library, respectively, toobtain final library sizes of 4.2×10¹⁰ for DP47-3 and 3.3×10⁹ forDP88-3. Phagemid particles displaying the Fab libraries were rescued andpurified by PEG/NaCl purification to be used for selections.

Example 3

Selection of Anti-FAP Clones (Primary Selections)

Selections were carried out against the ectodomain of human or murinefibroblast activating protein (FAP) which were cloned upstream apoly-lysine and a 6×his-tag. See SEQ ID NOs: 317 and 319. Prior toselections, the antigens were coated into immunotubes at a concentrationof either 10 μg/ml or 5 μg/ml, depending on round of selection.Selections were carried out according to the following protocol: (i)binding of ˜10¹² phagemid particles of library DP47-3 to immobilizedhuman or murine FAP for 2 hours; (ii) washing of immunotubes using 5×5mL PBS/Tween20 and 5×5 ml PBS; (iii) elution of phage particles byaddition of 1 mL 100 mM TEA (triethylamine) for 10 minutes andneutralization by the addition of 500 μL 1M Tris/HCl pH 7.4; and (iv)re-infection of log-phase E. coli TG1 cells, infection with helperphageVCSM13 and subsequent PEG/NaCl precipitation of phagemid particles to beused in subsequent selection rounds.

Selections have been carried out over three or four rounds usingdecreasing antigen concentrations of human FAP and in some cases usingmurine FAP at 5 μg/ml in the final selection round. Specific binderswere defined as signals 5× higher than background and were identified byELISA. NUNC maxisorp plates were coated with 10 μg/ml of human or murineFAP followed by addition of Fab-containing bacterial supernatants anddetection of specifically binding Fabs via their Flag-tags by using ananti-Flag/HRP secondary antibody.

ELISA-positive clones were bacterially expressed as 1 mL cultures in96-well format and supernatants were subjected to a kinetic screeningexperiment using BIACORE T100. K_(D) was measured by surface plasmonresonance using a BIACORE® T100 machine (GE Healthcare) at 25° C. withanti-human F(ab′)2 fragment specific capture antibody (JacksonImmunoResearch #109-005-006) immobilized by amine coupling on CM5 chipsand subsequent capture of Fabs from bacterial supernatant or frompurified Fab preparations. Briefly, carboxymethylated dextran biosensorchips (CM5, GE Healthcare) were activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Anti-human F(ab′)2 fragment specific capture antibody was diluted with10 mM sodium acetate, pH 5.0 at 50 μg/ml before injection at a flow rateof 10 μl/minute to achieve approximately up to 10.000 response units(RU) of coupled capture antibody. Following the injection of the captureantibody, 1 M ethanolamine was injected to block unreacted groups. Forkinetic measurements, Fabs from bacterial supernatant or purified Fabswere injected at a flow rate of 10 μl/min for 300 s and a dissociationof 300 s for capture baseline stabilization. Capture levels were in therange of 100-500 RU. In a subsequent step, human or murine FAP analytewas injected either as a single concentration or as a concentrationseries (depending of clone affinity in a range between 100 nM and 250pM) diluted in HBS-EP+ (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mMEDTA, 0.05% Surfactant P20, pH 7.4) at 25° C. at a flow rate of 50μl/min. Association time was 120 or 180 s, dissociation time was 300 to600 s. The surface of the sensorchip is regenerated by injection ofglycine pH 1.5 for 30 s at 90 μl/min followed by injection of NaOH for20s at the same flow rate. Association rates (k_(on)) and dissociationrates (k_(off)) were calculated using a simple one-to-one Langmuirbinding model (BIACORE® T100 Evaluation Software or Scrubber software(BioLogic)) by simultaneously fitting the association and dissociationsensorgrams. The equilibrium dissociation constant (K_(D)) wascalculated as the ratio k_(off)/k_(on).

Example 4

Construction of Anti-FAP Affinity Maturation Libraries

Three affinity maturation libraries were constructed on basis ofpre-selected antibodies from the primary anti-FAP selections. Moreprecisely, they were based on (i) anti-human FAP clone 2D9 (librarya.m.FAP2D9) (see SEQ ID NOs: 229 and 231), (ii) anti-murine FAP clone4B8 (library a.m.FAP4B8) (see SEQ ID NOs: 233 and 235) and (iii)cross-reactive clones 7A1, 13B2, 13C2, 13E8, 14C10 and 17A11 (librarya.m.FAPpool) (see SEQ ID NOs: 237 and 239 corresponding to the variableregion sequences of 7A1; SEQ ID NOs: 241 and 243 corresponding to thevariable region sequences of 13C2; SEQ ID NOs: 245 and 247 correspondingto the variable region sequences of 13E8; SEQ ID NOs: 249 and 251corresponding to the variable region sequences of 14C10; and SEQ ID NOs:253 and 255 corresponding to the variable region sequences of 17A11).

Each of these libraries consists of two sublibraries, randomized ineither CDR1 and CDR2 of the light chain (L1/L2) or CDR1 and CDR2 of theheavy chain (H1/H2), respectively. These sublibraries were pooled upontransformation. Each of these sublibraries was constructed by foursubsequent steps of amplification and assembly.

For L1/L2 libraries, the amplification and assembly protocol included:(i) amplification of fragment 1 (LMB3-DPK22_CDR1_rand ba_opt) andfragment 2 (DPK22_CDR1_fo-DPK22_Ck_BsiWI_ba); (ii) assembly of fragments1 and 2 using outer primers LMB3 and DPK22_Ck_BsiWI_ba to create thetemplate for fragment 3; (iii) amplification of fragment 3(LMB3-DPK22_CDR2_rand_ba) and fragment 4(DPK22_CDR2_fo-DPK22_Ck_BsiWI_ba); and (iv) final assembly of fragments3 and 4 using the same outer primers as above. See Table 5 for primersequences.

TABLE 5 SEQ Primers Used in L1/L2 Affinity Maturation IDLibraries for anti-FAP Affinity Maturation NO LMB3CAGGAAACAGCTATGACCATGATTAC 332 DPK22_CDR1_rand_CAGGTTTCTGCTGGTACCAGGCTAAG TA G C 342 ba_opt T G CT GCTAACACTCTGACTGGCCCTGCAAG DPK22_CDR1_fo TTAGCCTGGTACCAGCAGAAACCTG 343.DPK22_Ck_BsiWI_ GGTGCAGCCACCGTACGTTTGATTTCC 344 ba DPK22_CDR2_CTGTCTGGGATGCCAGTGGCCCTG CT G GA G 345 rand_ba GC G CC ATAGATGAGGAGCCTGGGAGCCTG DPK22_CDR2_fo AGGGCCACTGGCATCCCAGACAG 346 Bold:60% original base and 40% randomization as M Underline: 60% originalbase and 40% randomization as N

For H1/H2 libraries, the amplification and assembly protocol included:(i) amplification of fragment 1 (RJH53-DP47_CDR1_rand_ba_opt) andfragment 2 (DP47_CDR1_fo-MS52); (ii) assembly of fragments 1 and 2 usingouter primers RJH53 and MS52 to create the template for fragment 3;(iii) amplification of fragment 3 (RJH53-DP47_CDR2_rand_ba) and fragment4 (DP47_CDR2_fo-MS52); and (iv) final assembly of fragments 3 and 4using the same outer primers as above. See Table 6 for primer sequences.

TABLE 6 SEQ Primers Used in H1/H2 Affinity Maturation IDLibraries for anti-FAP Affinity Maturation NO RJH53CATCAGGGCCTGAGCTCGCCCGTCAC 347 DP47_CDR1_rand_ GAGCCTGGCGGACCCAGCTCATGGC A TA A C 348 ba_opt TGCTAAAGGTGAATCCGGAGGC DP47_CDR1_foATGAGCTGGGTCCGCCAGGCTC 349 MS52 GAAGACCGATGGGCCTTTGGTGCTAG 350DP47_CDR2_rand_ CCTTCACGGAGTCTGCGTAGTATGTG CT A C 351 ba C A CC A CT ACC ACTAATA GCTGAGACCCACT CCAGCCCCTTCCC DP47_CDR2_foACATACTACGCAGACTCCGTGAAGG 352 Bold: 60% original base and 40%randomization as M Underline: 60% original base and 40% randomization asN

Final assembly products have been digested with NcoI/BsiWI for L1/L2sublibraries of a.m.FAP2D9 and a.m.FAP4B8, with MunI and NheI for H1/H2sublibraries of a.m.FAP2D9 and a.m.FAP4B8 as well as with NcoI/BamHI forL1/L2 library of a.m.FAPpool and with BspEI/PstI for H1/H2 libraries ofa.m.FAPpool, respectively, alongside with similarly treated acceptorvectors based on plasmid preparations of clones 2D9, 4B8 or an equimolarmixture of clones 7A1, 13B2, 13C2, 13E8, 14C10 and 17A11, respectively.The following amounts of digested randomized (partial) V-domains anddigested acceptor vector(s) were ligated for the respective libraries(μg V-domain/m vector): a.m.FAP2D9 L1/L2 sublibrary (5.7/21.5),a.m.FAP2D9 H1/H2 sublibrary (4.1/15.5), a.m.FAP4B8 L1/L2 sublibrary(6.5/24.5), a.m.FAP4B8 H1/H2 sublibrary (5.7/21.5), a.m.FAPpool L1/L2sublibrary (4.4/20), a.m.FAPpool H1/H2 sublibrary (3.4/15.5).

Purified ligations of L1/L2 and H1/H2 sublibraries were pooled and usedfor 60 transformations for each of the 3 affinity maturation libraries,to obtain final library sizes of 6.2×10⁹ for a.m.FAP2D9, 9.9×10⁹ fora.m.FAP4B8 and 2.2×10⁹ for a.m.FAPpool.

Phagemid particles displaying these Fab libraries were rescued andpurified by PEG/NaCl purification to be used for secondary selections

Construction of Additional Anti-FAP Affinity Maturation Libraries (Basedon Clones 3F2, 3D9, 4G8, 4B3 and 2C6)

Four additional affinity maturation libraries were constructed on thebasis of pre-selected cross-reactive antibodies from the firstaffinity-maturation campaign of anti-FAP antibodies, namely clones 3F2,3D9, 4G8, 4B3 and 2C6 (see SEQ ID NOs: 195 and 197 corresponding to thevariable region sequences of 3F2; SEQ ID NOs: 199 and 201 correspondingto the variable region sequences of 3D9; SEQ ID NOs: 205 and 207corresponding to the variable region sequences of 4G8; SEQ ID NOs: 209and 211 corresponding to the variable region sequences of 4B3; SEQ IDNOs: 217 and 219 corresponding to the variable region sequences of 2C6).More precisely, the four libraries were based on 1) anti-FAP clones 3F2,4G8 and 4B3 (V_(H) library, randomized in CDRs 1 and 2 of variable heavychain, i.e. H1/H2 library), 2) anti-FAP clones 3D9 and 2C6 (V_(L)library, randomized in CDRs 1 and 2 of variable light chain, i.e. L1/L2library), 3) anti-FAP clone 3F2 (L3 library with soft randomization inCDR3 of light chain, i.e. L3 library) and 4) anti-FAP clone 3F2 (H3library with soft randomization in CDR3 of heavy chain, i.e. H3library). The first two libraries were constructed exactly the same wayas outlined for the first affinity-maturation campaign of anti-FAPantibodies, for the L1/L2 and H1/H2 libraries, respectively. Incontrast, for the L3 and H3 affinity-maturation libraries based on clone3F2, two new primers were used to introduce soft randomization in L3(AM_(—)3F2_DPK22_L3_ba:

CACTTTGGTCCCCTGGCCGAACGT CGGGGGAAGCA TAATACCCTGCTGACAGTAATACACTGCwith underlined bases being 60% given base and 40% mixture N (mixture ofthe four nucleotides A, C, G, and T)) and H3 (AM_(—)3F2_DP47_H3_fo:

GGCCGTATATTACTGTGCG AAA GGG TGG TTT  GGT GGT TTT AAC TACTGGGGCCAAGGAACwith underlined bases being 60% given base and 40% mixture N, bases initalics being 60% given base and 40% G, as well as underlined bases initalics being 60% given base and 40% mixture K (mixture of the twonucleotides G and T)) of the parental clone. Library sizes were asfollows: H1/H2 library (1.13×10¹⁰), L1/L2 library (5.6×10⁹), L3 library(2.3×10¹⁰) and H3 library (2.64×10¹⁰).

Example 5

Selection of Affinity-Matured Anti-FAP Clones

Selections were carried out against the ectodomain of human or murinefibroblast activating protein (FAP) which were cloned 5′ of apoly-lysine and a 6×his-tag. See SEQ ID NOs: 317 and 319. Prior toselections, the antigens were coated into immunotubes at a concentrationof either 10 μg/mL, 5 μg/mL or 0.2 μg/mL, depending on the library andround of selection. Selections were carried out according to thefollowing protocol: (i) binding of ˜10¹² phagemid particles of librarya.m.FAP2D9, a.m.FAP4B8 or a.m.FAPpool to immobilized human or murine FAPfor 2 hours; (ii) washing of immuno tubes using 10−20×5 mL PBS/Tween20and 10−20×5 mL PBS (depending on library and selection round); (iii)elution of phage particles by addition of 1 mL 100 mM TEA(triethylamine) for 10 minutes and neutralization by addition of 500 μL1M Tris/HCl pH 7.4; and (iv) re-infection of log-phase E. coli TG1cells, infection with helperphage VCSM13 and subsequent PEG/NaClprecipitation of phagemid particles to be used in subsequent selectionrounds.

Selections were carried out over 2 rounds and conditions were adjustedfor each of the 3 libraries individually. In detail, selectionparameters were: a.m.FAP2D9 (5 μg/mL human FAP and 20 washes in totalfor round 1, 1 μg/mL human FAP and 30 washes in total for round 2),a.m.FAP4B8 (1 μg/mL murine FAP and 30 washes in total for round 1, 0.2μg/mL human FAP and 40 washes in total for round 2) and a.m.FAPpool (5μg/mL human FAP and 30 washes in total for round 1, 5 μg/mL murine FAPand 30 washes in total for round 2). Specific binders were defined assignals 5× higher than background and were identified by ELISA. NUNCmaxisorp plates were coated with 1 μg/mL or 0.2 μg/mL of human or murineFAP followed by addition of Fab-containing bacterial supernatants anddetection of specifically binding Fabs via their Flag-tags by using ananti-Flag/HRP secondary antibody.

ELISA-positive clones were bacterially expressed as 1 ml cultures in96-well format and supernatants were subjected to a kinetic screeningexperiment using BIACORE T100, as described above (see Example 3).

Additional Selection of Affinity-Matured Anti-FAP Clones

Selections were carried out against the ectodomain of human and murinefibroblast activating protein (FAP) which were cloned upstream a6×-lysine and a 6×-his tag (see SEQ ID NOs: 317 and 319). Prior toselections, the antigens were coated into immunotubes at a concentrationof either 1 μg/ml, 0.2 μg/ml or 0.02 μg/ml, depending on the library andround of selection.

Selections and ELISA-based screenings were carried out as described forthe first affinity-maturation campaign of anti-FAP antibodies. Secondaryscreenings were carried out using a ProteOn XPR36 biosensor (Biorad),and kinetic rate constants and affinities were determined analyzingaffinity-purified Fab preparations on the same instrument. K_(D) wasmeasured by surface plasmon resonance using a ProteOn XPR36 machine(Biorad) at 25° C. with anti-human F(ab′)2 fragment specific captureantibody (Jackson ImmunoResearch #109-005-006) immobilized on GLM chipsand subsequent capture of Fabs from bacterial supernatant or frompurified Fab preparations. Briefly, GLM biosensor chips (Biorad) wereactivated for 5 min with a freshly prepared mixture ofN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS). Anti-human F(ab′)2 fragment specific captureantibody was diluted to 24 μg/ml with 10 mM sodium acetate, pH 5.0before injection for 5 min to achieve approximately up to 10.000response units (RU) of coupled capture antibody. Following the injectionof the capture antibody, 1 M ethanolamine was injected for 5 min toblock unreacted groups. For kinetic measurements, Fabs from bacterialsupernatant were injected at a flow rate of 30 μl/min for 100 s. Capturelevels were in the range of 250 RU. In a subsequent step, serialdilutions of human, murine or cynomolgus FAP analyte were injected(two-fold dilution, highest concentration 25 nM) diluted in PBS/0.005%Tween-20 at 25° C. at a flow rate of 50 μl/min. Association time was 240s, dissociation time 600 to 1800 s. The sensorchip was regenerated byinjection of 0.85% H₃PO₄ for 30 s at 100 μl/min followed by injection of50 mM NaOH for 30 s at the same flow rate. Association rates (k_(on))and dissociation rates (k_(off)) were calculated using a simpleone-to-one Langmuir binding model (ProteOn manager software version 2.1)by simultaneously fitting the association and dissociation sensorgrams.The equilibrium dissociation constant (K_(D)) is calculated as the ratiok_(off)/k_(on).

The following affinity-matured clones were identified: 19G1 (see SEQ IDNOs: 257 and 259), 20G8 (see SEQ ID NOs: 281 and 263), 4B9 (see SEQ IDNOs: 265 and 267), 5B8 (see SEQ ID NOs: 269 and 271), 5F1 (see SEQ IDNOs: 273 and 275), 14B3 (see SEQ ID NOs: 277 and 279), 16F1 (see SEQ IDNOs: 281 and 283), 16F8 (see SEQ ID NOs: 285 and 287), O3C9 (see SEQ IDNOs: 289 and 291), 22A3 (see SEQ ID NOs: 301 and 303) and 29B11 (see SEQID NOs: 305 and 307) (all these clones were selected from the H1/H2library and are derived from parental clone 3F2), O2D7 (see SEQ ID NOs:293 and 295) (selected from the L3 library based on parental clone 3F2),and 28H1 (see SEQ ID NOs: 297 and 299) and 23C10 (see SEQ ID NOs: 309and 311) (these two clones were selected from the H1/H2 library and arederived from parental clone 4G8).

FIGS. 1A-F, 2A-F, 3A-F, 4A-H, and 5A-F show the Surface PlasmonResonance sensorgrams of selected affinity matured Fabs binding toimmobilized FAP and Table 7 gives the respective affinities derived. Theselected Fabs span a high affinity range in the pM to nM range and arecross-reactive for human (hu) and murine (mu) FAP, as well as Cynomolgus(cyno) FAP as determined for selected clones. The affinity maturedanti-FAP Fabs were converted into the Fab-IL2-Fab format and into IgGantibodies for specificity analysis. Specificity of binding was shown bylack of binding to DPPIV as close homologue of FAP, expressed on HEK293or CHO cells (see Example 9).

TABLE 7 Summary of kinetic equilibrium constants (K_(D)) ofaffinity-matured anti-FAP antibodies as Fab fragments (monovalentbinding). affinity (K_(D)) to affinity (K_(D)) to affinity (K_(D)) toantibody hu FAP [pM] mu FAP [pM] cyno FAP [pM] 19G1 76 2600 n.d. 20G8 692800 n.d. 4B9 157 3300 n.d. 5B8 690 3200 n.d. 5F1 243 4100 n.d. 14B3 3773800 n.d. 16F1 193 3400 n.d. 16F8 301 3800 n.d. O3C9 160 3700 n.d. O2D7619 8300 n.d. 28H1 200 9 3600 22A3 34 655 522 29B11 35 436 23 23C10 1600125 990

Example 6

IgG Conversion of Fabs Binding FAP

The parental 3F2, 4G8 and 3D9 Fabs and the affinity matured 3F2 and 4G8Fab derivatives have been converted into a human IgG1 format, a mouseIgG2a format and a human IgG1 format. The full antibody heavy and lightchain DNA sequences were obtained either by subcloning the variableregions in frame with the respective constant heavy and the constantlight chain regions pre-inserted into different recipient mammalianexpression vectors or were recombined by fusing a short sequence stretchhomologous to the recipient vectors insertion site. The recombinationwas performed according to the “In-Fusion Cloning System” fromInvitrogen.

In all vectors the antibody expression is driven by an MPSV promoter andall vectors carry a synthetic polyA signal sequence at the 3′ end of theCDS. In addition each vector contains an EBV OriP sequence.

Example 7

Biacore Analysis of Anti-FAP IgG antibodies

The affinity of the anti-FAP Fab fragments 3F2, 4G8 and 3D9 as well asof the human IgG1 converted anti-FAP antibodies was subsequentlydetermined and confirmed for human, murine and Cynomolgus FAP by SurfacePlasmon Resonance (SPR) analysis at 25° C. using a BIACORE® T100 machine(GE Healthcare). For this purpose, human, mouse or Cynomolgus FAPextracellular domain (SEQ ID NOs 317-322) was captured by an immobilizedanti-His antibody (Penta His Qiagen 34660) and the antibodies were usedas analytes. For immobilization carboxymethylated dextran biosensorchips (CMS, GE Healthcare) were activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions. ThePenta His antibody was diluted with 10 mM sodium acetate, pH 5, to 40μg/ml before injection at a flow rate of 10 μl/minute to achieveapproximately 9000 response units (RU) of coupled protein. Following theinjection of the ligand, 1 M ethanolamine was injected to blockunreacted groups.

For kinetics measurements, human, mouse or Cynomolgus FAP extracellulardomain was injected at 10 μl/min at 10 nM for 20 s (for Fab fragments)or at 20 nM for 25 s (for IgG) and was captured via its His tag by theimmobilized penta His antibody. Serial dilutions of antibody (two-folddilutions, range between 6.25 nM to 200 nM for Fab fragments orfive-fold dilutions, range between 3.2 pM to 10 nM for IgG) wereinjected in HBS-EP+ (GE Healthcare, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA,0.05% Surfactant P20, pH 7.4) at 25° C. at a flow rate of 90 μl/min. Thefollowing parameters were applied: Association time 180 s, dissociation300 s (for Fab) or 900 s (for IgG), regeneration with 10 mM glycine pH 2for 60 s between each cycle. Association rates (k_(on)) and dissociationrates (k_(off)) were calculated using a simple one-to-one Langmuirbinding model (BIACORE® T100 Evaluation Software version 1.1.1) bysimultaneously fitting the association and dissociation sensorgrams(model parameters were local Rmax and RI=0). The equilibriumdissociation constant (K_(D)) was calculated as the ratiok_(off)/k_(on).

The K_(D) values of binding are given in Table 8. FIGS. 6A1-C3 shows thecorresponding SPR-based kinetic analyses for Fab fragments, FIGS. 7A1-C3for IgG antibodies.

TABLE 8 Summary of kinetic equilibrium constants (K_(D)) of 3F2, 4G8 and3D9 anti-FAP antibodies as Fab fragments and as IgG Construct Human FAPMurine FAP Cyno FAP IgG 3F2 Avidity: 39 pM Avidity: 29 pM Avidity: 42 pMIgG 4G8 Avidity: 51 pM Avidity: 1 pM Avidity: 59 pM IgG 3D9 Avidity: 93pM Avidity: 96 pM Avidity: 96 pM Fab fragment 3F2 Affinity: 13 nMAffinity: 14 nM Affinity: 11 nM Fab fragment 4G8 Affinity: 74 nMAffinity: 7 nM Affinity: 56 nM or lower Fab fragment 3D9 Affinity: 133nM Affinity: 32 nM Affinity: 143 nM

Example 8

Binding of Anti-FAP Antibodies 3F2, 4G8 and 3D9 on Human Tumor TissueSections

We performed experiments to detect and compare the expression of FAP infresh frozen human tumor tissues (breast cancer, colon adenocarcinomasand NSCLC tissues) using the anti-FAP antibodies clones 3F2, 4G8 and 3D9as mouse IgG2a.

One fresh frozen tissue microarray (TMA) (AST 274), containing thirtydifferent tumors with two spots each, was used from the Roche TRSPathology & Tissue Biomarkers tumorbank. The TMA containing 10 invasiveductal carcinomas of the breast, 10 colorectal adenocarcinomas and 10non-small cell lung cancers was obtained from Asterand Ltd, Royston, UK.

For the immunohistochemical (IHC) stainings, the following antibodieswere used: monoclonal mouse anti-human FAP clone 3F2 (15.8 ng/ml,diluted in Ventana Antibody Diluent), monoclonal mouse anti-human FAPclone 4G8 (1000 ng/ml, diluted in Ventana Antibody Diluent), andmonoclonal mouse anti-human FAP clone.3D9 (1000 ng/ml, diluted inVentana Antibody Diluent). A polyclonal mouse IgG2a, concentration 100μg/mL (Provider: DAKO, X0943, lot #00058066) was used as isotypecontrol.

The stainings were performed according to standard protocols on aVentana Benchmark XT instrument, using the Ventana Ultra-View detectionkit with HRP-system for detection (containing Universal HRP Multimer,and DAB for staining) Counter-staining was done with Hematoxylin II(Ventana, Mayer's Hematoxylin) and Blueing Reagent (Ventana) for 8 min.

The TMA was analyzed semi-quantitatively and the total FAP expression(staining intensity) as well as the localization of the FAP expressionin the tumor tissue was evaluated.

With all three anti-FAP antibodies, all the tumor tissue samples (breastcancer, colorectal cancer and lung cancer) that could be evaluatedshowed a moderate to strong staining FAP signal intensity in the stromacomponent of the tumor. At least 7 out of 10 samples for each tumor andantibody could be evaluated. The remaining samples could not beevaluated, because tissue cores had folding artifacts, contained onlynormal tissue, or were missing.

As expected, the FAP signal was invariably located in the. stromacomponent of tumors. There was a slight difference in signal intensitybetween clone 3F2 and clones 3D9 and 4G8. A slightly stronger signal wasseen with clones 3D9 and 4G8, the difference was minor, however.

FIGS. 8A1-D2 shows representative micrographs of human tumor tissuesamples immunohistochemically stained for FAP using the anti-FAP mouseIgG2a 3F2, 3D9 or 4G8, or an isotype control antibody.

Example 9

Binding of Anti-FAP Antibodies to FAP on Cells

Binding of human IgG1 antibodies 3F2, 4B3 and 4G8 to human and murineFAP expressed on stably transfected HEK293 cells was measured by FACS.Briefly, 150.000 cells per well were incubated with the indicatedconcentration of the anti-FAP antibodies 3F2, 4B3 and 4G8 in a roundbottom 96-well plate, incubated for 30 min at 4° C., and washed oncewith PBS/0.1% BSA. Bound antibody was detected with FITC-conjugatedAffiniPure F(ab′)2 Fragment goat anti-human F(ab′)2 Specific (JacksonImmuno Research Lab #109-096-097, working solution: 1:20 diluted inPBS/0.1% BSA, freshly prepared) after incubation for 30 min at 4° C.using a FACS Cantoll (Software FACS Diva). The results are shown inFIGS. 9A and 9B. EC50 values at half-maximal binding for binding tohuman and murine FAP were determined and are given in Table 9.

TABLE 9 Binding of anti-FAP antibodies to FAP on cells (EC50 values).EC50 values on cells [nM] human FAP murine FAP 3F2 IgG 4.8 1.0 4B3 IgG5.5 1.6 4G8 IgG 5.0 1.7

Specificity of FAP Antibodies

In order to assess the specificity of binding of the phage displayderived antibodies, binding to HEK293 cells stably expressing DPPIV (aclose homologue of FAP that is expressed on healthy tissues) or HER2 wasmeasured for the anti-FAP human IgG1 antibodies 3F2, 4B3 and 4G8.

Briefly, 200.000 cells per well (HEK293-DPPIV or HEK293-HER2 as control)were incubated with 30 μg/ml of the anti-FAP antibodies 3F2, 4B3 or 4G8in a round bottom 96-well plate, incubated for 30 min at 4° C. andwashed once with PBS/0.1% BSA. Trastuzumab (anti-HER2 antibody) or aphycoerythrin (PE)-conjugated mouse anti-human anti-CD26/DPPIV antibody(CD26=DPPIV, mouse IgGl,k, BD Biosciences, #555437, clone M-A261) wereused as positive controls. Bound antibody was detected withPE-conjugated AffiniPure F(ab′)2 Fragment goat anti-human IgG FcγSpecific (Jackson Immuno Research Lab #109-116-170, working solution:1:20 diluted in PBS/0.1% BSA, freshly prepared) after incubation for 30min at 4° C. using a FACS Cantoll (Software FACS Diva). The results ofthis experiment are shown in FIG. 10. None of the anti-FAP antibodiesshowed significant binding to DPPIV or HER2, but signals in the range ofthe negative controls (secondary antibody alone, isotype controlantibody, or no antibody at all).

Binding of Anti-FAP Antibodies to FAP on Human Fibroblasts

Binding of human IgG1 antibodies to human FAP expressed on humanfibroblast cell line GM05389 (derived from human fetal lung, NationalInstitute of General Medical Sciences, Camden, N.J.) was measured byFACS. Briefly, 200.000 cells per well were incubated with 30 μg/ml ofthe anti-FAP antibodies 3F2 or 4G8 in a round bottom 96-well plate,incubated for 30 min at 4° C. and washed once with PBS/0.1% BSA. Boundantibody was detected with FITC-conjugated AffiniPure F(ab′)2 Fragmentgoat anti-human IgG Fcγ Specific (Jackson Immuno Research Lab#109-096-098, working solution: 1:20 diluted in PBS/0.1% BSA, freshlyprepared) after incubation for 30 min at 4° C. using a FACS Cantoll(Software FACS Diva). The results of this experiment are shown in FIGS.11A-D. Both anti-FAP antibodies strongly bind to FAP expressed on humanfibroblasts.

Binding of Anti-FAP Antibodies to FAP on Human Tumor Cells

Binding of human IgG1 antibodies to human FAP expressed on humanfibroblasts cell line GM05389 and on stably transfected HEK293 cells wascompared to FAP expression on human cancer cell lines ACHN, Colo205,MDA-MB231, MDA-MB435 and KPL4 by FACS.

Briefly, 200.000 cells per well were incubated with 10 μg/ml of theanti-FAP antibodies 3F2 or 4G8 in a round bottom 96-well plate,incubated for 30 min at 4° C. and washed once with PBS/0.1% BSA. Boundantibody was detected with FITC-conjugated AffiniPure F(ab′)2 Fragmentgoat anti-human F(ab′)2 Specific (Jackson Immuno Research Lab#109-096-097, working solution: 1:20 diluted in PBS/0.1% BSA, freshlyprepared) after incubation for 30 min at 4° C. using a FACS Cantoll(Software FACS Diva). The results of this experiment are shown in FIG.12.

The data show that the antibodies 3F2 and 4G8 bind specifically to FAPthat is strongly overexpressed on fibroblasts and stably transfectedHEK293 cells; whereas only weak binding can be detected on ACHN,Colo205, MDA-MB231, MDA-MB435 and KPL4 human tumor cell lines.

Example 10

Analysis of FAP Internalization upon Binding of Anti-FAP Antibody byFACS

For several FAP antibodies known in the art it is described that theyinduce FAP internalization upon binding (described e.g. in Baum et al.,J Drug Target 15, 399-406 (2007); Bauer et al., Journal of ClinicalOncology, 2010 ASCO Annual Meeting Proceedings (Post-Meeting Edition),vol. 28 (May 20 Supplement), abstract no. 13062 (2010); Ostermann etal., Clin Cancer Res 14, 4584-4592 (2008)).

Thus, we analyzed the internalization properties of our antibodies.Briefly, GM05389 cells (human lung fibroblasts,) cultured in EMEM medium+15% FCS, were detached, washed, counted, checked for viability andseeded at a density of 0.2 mio cells/well in 12 well plates. The nextday, FAP antibodies 4G8 and 3F2 (FIG. 13A) or 4G8 only (FIG. 13B) werediluted to 10 μg/ml in cold medium, cells were cooled down on ice andthe diluted antibodies (0.5 ml/well) or medium alone were added asindicated. Subsequently, cells were incubated for 30 min in the coldroom with gentle agitation, followed by addition of 0.5 ml warm mediumand further incubation of the cells at 37° C. for the indicated timeperiods. When the different time points were reached, cells weretransferred to ice, washed once with cold PBS and incubated with 0.4 mlof the secondary antibody (Alexa Fluor 633-conjugated goat anti-humanIgG, Molecular Probes #A-21091, 2 mg/ml, use 1:500) for 30 min at 4° C.Cells were then washed twice with PBS/0.1% BSA, transferred to a 96 wellplate, centrifuged for 4 min at 4° C., 400×g and cell pellets wereresuspended by vortexing. Cells were fixed using 100 μl 2% PFA. For FACSmeasurement, cells were re-suspended in 200 μl/sample PBS/0.1% BSA andmeasured with the plate protocol in FACS Cantoll (Software FACS Diva).The results of these experiments are presented in FIGS. 13A and B, andshow that the 4G8 and 3F2 anti-FAP antibodies do not induceinternalization of FAP on fibroblasts.

Analysis of FAP Internalization upon Binding of Anti-FAP Antibody byImmunofluorescence

GM05389 cells (human lung fibroblasts) were grown on glass coverslips inEMEM medium+15% FCS. Before treatment, cells were washed three timeswith PBS and starved in EMEM medium+0.1% BSA for 2 h. The anti-FAPantibody (4G8 IgG) or an anti-CD20 antibody (GA101, used as isotypecontrol) were diluted in cold EMEM medium to the final concentration of10 μg/ml. After starvation, cells were cooled on ice, rinsed twice withcold PBS and incubated with the diluted antibodies (0.5 ml/well) for 45min at 4° C. under constant agitation to allow surface binding. Cellswere then washed twice with cold PBS and either fixed with cold PFA (T0,paraformaldehyde 4% in PBS pH 7.4) or further incubated at 37° C. for 20min, 1 h, 3 h and 6 h in EMEM+10% FCS. At each time point, cells werewashed twice with cold PBS and PFA-fixed for 20 min on ice. Afterfixation, cells were washed four times with cold PBS, permeabilized withTriton 0.03% and incubated with anti-EEA1 (early endosome marker)antibody for 45 min at room temperature in blocking buffer (PBS+10%FCS). Cells were then washed three times with PBS and incubated withfluorescently labeled secondary antibodies (donkey anti-mouse AlexaFluor 594-conjugated antibody, and goat anti-human Alexa Fluor488-conjugated antibody) at room temperature for further 45 min. Cellswere finally washed and mounted on glass support slides using ImmunoMount mounting medium.

FIGS. 14A1-D4 presents representative immunofluorescence images showingFAP plasma membrane staining on GM05389 lung fibroblasts obtained afterbinding of anti-FAP 4G8 IgG for 45 min at 4° C. (A1), for 20 min at 37°C. (B1), for 1 hour at 37° C. (C1) or for 6 hours at 37° C. (D1). Theanti-CD20 antibody GA101, used as isotype control, shows backgroundstaining (A3, B3, C3, D3). EEA1 labels early endosomes(A2, B2, C2, D2;GA101 isotype control is shown in A4, B4, C4, D4)). Note the persistenceof the FAP surface plasma membrane staining up to 6 hours after anti-FAP4G8 antibody binding.

Example 11

Biacore Analysis of Affinity-Matured Anti-FAP IgG Antibodies

Affinity matured anti-FAP Fab fragments derived from 3F2 and 4G8 wereconverted into rabbit IgG antibodies. The affinity of the affinitymatured 3F2 and 4G8-based rabbit IgG1 converted anti-FAP antibodies toFAP is subsequently determined and confirmed for human, murine andCynomolgus FAP by SPR analysis at 25° C. (Biacore). For this purpose,human, mouse or Cynomolgus FAP extracellular domain (SEQ ID NOs 317-322)is captured by an immobilized anti-His antibody (Penta His Qiagen 34660)and the antibodies are used as analytes. IgGs are diluted 1:5 from 10 nMto 3.2 pM. The following parameters are applied: Association time 180 s,dissociation 900 s, flow 90 μl/min. Regeneration with 10 mM glycine pH 2for 60 s. The curves were fitted with the 1:1 model to get the K_(D)values (Rmax local, RI=0).

Example 12

Binding of Affinity Matured Anti-FAP Antibodies to FAP on Cells

Binding of affinity matured human IgG1 antibody 28H1 labeled withAlexa-647 (1.89 mg/ml, 1.83 mole dye/mole protein) derived from 4G8parental antibody to human FAP expressed on stably transfected HEK293cells was measured by FACS. Briefly, 200.000 cells per well wereincubated with the indicated concentration of 2 μg/ml and 10 μg/ml ofthe parental 4G8 and affinity matured 28H1 anti-FAP antibodies in around-bottom 96-well plate, incubated for 30 min at 4° C. and washedonce with PBS/0.1% BSA. Bound antibody was detected after incubation for30 min at 4° C. using a FACS Cantoll (Software FACS Diva). The data showthat both antibodies bind strongly to HEK293 cells transfected withhuman FAP (FIG. 23).

Example 13

Binding of Affinity Matured Anti-FAP Antibodies to FAP on HumanFibroblasts

Binding of affinity matured human IgG1 antibodies derived from 3F2 tohuman FAP expressed on human fibroblast cell line GM05389 (derived fromhuman fetal lung, National Institute of General Medical Sciences,Camden, N.J.) is measured by FACS. Briefly, 200.000 cells per well areincubated with 30 μg/ml of the affinity matured 3F2 anti-FAP antibody ina round-bottom 96-well plate, incubated for 30 min at 4° C. and washedonce with PBS/0.1% BSA. Bound antibody is detected with FITC-conjugatedAffiniPure F(ab′)2 Fragment goat anti-human IgG Fcγ Specific (JacksonImmuno Research Lab #109-096-098, working solution: 1:20 diluted inPBS/0.1% BSA, freshly prepared) after incubation for 30 min at 4° C.using a FACSCantoII (Software FACS Diva). EC50 values at half-maximalbinding for binding to human and murine FAP are being determined.

Example 14

Antibody-Dependent Cell-Mediated Cytotoxicity Mediated byGlycoengineered Anti-FAP IgG1 Antibodies

Human IgG1 antibodies against FAP derived from 4G8 or 3F2 wereglycoengineered by co- transfection with plasmids encoding for GnTIIIand ManII as described in Example 1.

Subsequently, glycoengineered parental 4G8 and 3F2 and affinity matured28H1 human IgG1 antibodies were compared in an ADCC assay for theirpotential to mediate superior antibody mediated cellular cytotoxicitycompared to their non-glycoengineered wildtype versions.

Briefly, HEK293 cells stably transfected with human FAP as target cellswere collected, washed and resuspended in culture medium, stained withfreshly prepared Calcein AM (Molecular Probes) at 37° C. for 30 min,washed three times, counted and diluted to 300.000 cells/ml. Thissuspension was transferred to a round-bottom 96-well plate (=30.000cells/well), the respective antibody dilution was added and incubatedfor 10 min to facilitate the binding of the tested antibody to the cellsprior to contact with effector cells. Effector to target ratio was 25 to1 for PBMCs. Co-incubation was performed for 4 hours. As readout therelease of lactate dehydrogenase (LDH) into supernatant afterdisintegration of the attacked cells was determined. LDH from co-culturesupernatant was collected and analyzed with a LDH detection Kit (RocheApplied Science). Substrate conversion by the LDH enzyme was measuredwith an ELISA absorbance reader (SoftMaxPro software, referencewavelengths: 490 nm versus 650 nm). As shown in FIG. 24 all anti-FAPantibodies tested were able to induce ADCC on HEK293-hFAP cells. Theglycoengineered (ge) versions performed always better than thecorresponding wildtype (wt) non-glycoengineered version.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

1-48. (canceled)
 49. A method of treating an individual having a diseasecharacterized by Fibroblast Activation Protein (FAP) expression,comprising administering to the individual an effective amount of anantibody that specifically binds to FAP, wherein said antibody comprises(a) a heavy chain CDR1 comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 3, SEQ ID NO: 13, and SEQ ID NO: 23;(b) a heavy chain comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 47, SEQ ID NO: 81, and SEQ ID NO: 113;(c) a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:135; (d) a light chain CDR1 comprising the amino acid sequence of SEQ IDNO: 143; (e) a light chain CDR2 comprising the amino acid sequence ofSEQ ID NO: 151; and (f) a light chain CDR3 comprising the amino acidsequence of SEQ ID NO:
 163. 50. The method of claim 49, wherein saidantibody comprises (i) a heavy chain variable region comprising theheavy chain CDR1 of SEQ ID NO: 3; the heavy chain CDR2 of SEQ ID NO: 47;and the heavy chain CDR3 of SEQ ID NO: 135, and a light chain variableregion comprising the light chain CDR1 of SEQ ID NO: 143, the lightchain CDR2 of SEQ ID NO: 151, and the light chain CDR3 of SEQ ID NO:163; (ii) a heavy chain variable region comprising the heavy chain CDR1of SEQ ID NO: 13; the heavy chain CDR2 of SEQ ID NO: 81; and the heavychain CDR3 of SEQ ID NO: 135, and a light chain variable regioncomprising the light chain CDR1 of SEQ ID NO: 143, the light chain CDR2of SEQ ID NO: 151, and the light chain CDR3 of SEQ ID NO: 163; or (iii)a heavy chain variable region comprising the heavy chain CDR1 of SEQ IDNO: 23; the heavy chain CDR2 of SEQ ID NO: 113; and the heavy chain CDR3of SEQ ID NO: 135, and a light chain variable region comprising thelight chain CDR1 of SEQ ID NO: 143, the light chain CDR2 of SEQ ID NO:151, and the light chain CDR3 of SEQ ID NO:
 163. 51. The method of claim49, wherein said antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO:
 267. 52. The method ofclaim 49, wherein said antibody comprises a light chain variable regioncomprising the amino acid sequence of SEQ ID NO:
 265. 53. The method ofclaim 49, wherein said antibody comprises a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 267 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 265.54. The method of claim 49, wherein said antibody comprises an Fc regionor a region equivalent to the Fc region of an immunoglobulin.
 55. Themethod of claim 54, wherein said Fc region is an IgG Fc region.
 56. Themethod of claim 49, wherein said antibody is a full-length IgG classantibody.
 57. The method of claim 49, wherein said antibody comprises ahuman constant region.
 58. The method of claim 49, wherein said antibodyis a human antibody.
 59. The method of claim 49, wherein said antibodycomprises a glycoengineered Fc region.
 60. The method of claim 59,wherein said antibody has an increased proportion of non-fucosylatedoligosaccharides in said Fc region, as compared to a non-glycoengineeredantibody.
 61. The method of claim 59, wherein at least 20% to 100% ofthe N-linked oligosaccharides in said Fc region are non-fucosylated. 62.The method of claim 59 wherein said antibody has an increased proportionof bisected oligosaccharides in said Fc region, as compared to anon-glycoengineered antibody.
 63. The method of claim 59, wherein atleast 20% to 100% of the N-linked oligosaccharides in said Fc region arebisected.
 64. The method of claim 59, wherein at least 20% to 50% of theN-linked oligosaccharides in said Fc region are bisected,non-fucosylated.
 65. The method of claim 49, wherein said antibody hasincreased effector function and/or increased Fc receptor bindingaffinity.
 66. The method of claim 65, wherein said increased effectorfunction is increased ADCC.
 67. The method of claim 49, wherein saidantibody is in a conjugate with a cytotoxic agent.
 68. The method ofclaim 49 further comprising administering an additional therapeuticagent to the individual.
 69. The method of claim 49, wherein saiddisease is cancer.
 70. The method of claim 69, wherein said cancer isselected from the group consisting of lung cancer, colon cancer, gastriccancer, breast cancer, head and neck cancer, skin cancer, liver cancer,kidney cancer, prostate cancer, pancreatic cancer, brain cancer, cancerof the skeletal muscle.
 71. The method of claim 49, wherein saidindividual is a human.